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Unhealthy diets contribute substantially to the worldwide burden of non-communicable diseases, such as cardiovascular diseases, cancers, and diabetes. Globally, non-communicable diseases are the leading cause of death, and numbers are still rising, which makes healthy diets a global priority. In Nutrition Research, two fields are particularly relevant for formulating healthier diets: dietary assessment, which assesses food and nutrient intake in order to investigate the relation between diet and disease, and dietary advice, which translates food and nutrient recommendations into realistic food choices. Both fields face complex decision problems: which foods to include in dietary assessment or advice in order to pursue the multiple objectives of the researcher or fulfil the requirements of the consumer. This thesis connects the disciplines of Nutrition Research and Operations Research in order to contribute to formulating healthier diets. In the context of dietary assessment, the thesis proposes a MILP model for the selection of food items for food frequency questionnaires (a crucial tool in dietary assessment) that speeds up the selection process and increases standardisation, transparency, and reproducibility. An extension of this model gives rise to a 0-1 fractional programming problem with more than 200 fractional terms, of which in every feasible solution only a subset is actually defined. The thesis shows how this problem can be reformulated in order to eliminate the undefined fractional terms. The resulting MILP model can solved with standard software. In the context of dietary advice, the thesis proposes a diet model in which food and nutrient requirements are formulated via fuzzy sets. With this model, the impact of various achievement functions is demonstrated. The preference structures modelled via these achievement functions represent various ways in which multiple nutritional characteristics of a diet can be aggregated into an overall indicator for diet quality. Furthermore, for Operations Research the thesis provides new insights into a novel preference structure from literature, that combines equity and utilitarianism in a single model. Finally, the thesis presents conclusions of the research and a general discussion, which discusses, amongst others, the main modelling choices encountered when using MODM methods for optimising diet quality. Summarising, this thesis explores the use of MODM approaches to improve decision-making for dietary assessment and advice. It provides opportunities for better decision-making in research on dietary assessment and advice, and it contributes to model building and solving in Operations Research. Considering the added value for Nutrition Research and the new models and solutions generated, we conclude that the combination of both fields has resulted in synergy between Nutrition Research and Operations Research.

Taste and smell changes are common side effects during chemotherapy in cancer patient and may have an impact on food preferences, food intake and quality of life. However, these relations have hardly been studied systematically in specific cancer populations. The overall aim of this thesis was to assess how the sense of taste and smell change upon treatment with chemotherapy in breast cancer and oesophagogastric cancer patients, and to investigate their consequences in terms of food preferences, food intake and quality of life. To measure food preferences for both macronutrients and tastes, the Macronutrient and Taste Preference Ranking Task (MTPRT) was developed. in chapter 2, it was shown that by inducing sensory specific satiety for a standardized sweet and savoury meal, it is possible to detect shifts in preferences for both tastes and macronutrients with the MTPRT, and that these results are reproducible. In Chapter 3 we studied objective and subjective taste and smell perception and food preferences in advanced oesophagogastric cancer patients undergoing palliative chemotherapy. The result showed that only objective taste function decreases during chemotherapy, but other chemosensory measures were unchanged. A lower subjective taste perception was related to a lower preference for high-protein products. Therefore it is important to consider patients’ taste perception, when providing dietary advice to OGC patients Chapter 4 describes a study with similar outcome measures as chapter 3, but in breast cancer patients at several time points during and after chemotherapy, and compared to a healthy control group. The study showed that breast cancer patients like high-protein, high-fat, sweet and savoury products less during chemotherapy, thus showing altered preferences for macronutrients, but not for tastes. Furthermore, results showed a temporary decrease in taste and smell perception during chemotherapy. These findings show that patients should be informed prior to treatment on chemosensory changes, and that these changes should be monitored during treatment due to the consequences for nutritional intake and quality of life In chapter 5 we assessed the dietary intake of breast cancer patients before and during chemotherapy compared to a healthy control group, and associations with experienced symptoms during chemotherapy. It was shown that symptoms induced by chemotherapy were associated with lower total energy, protein and fat intake, which was manifested by a lower intake of specific food groups. Therefore, to ensure an optimal dietary intake during chemotherapy, it is important to monitor nutritional status and symptom burden during chemotherapy in breast cancer patients. To better understand the impact of chemosensory changes during chemotherapy on daily life, 13 advanced oesophagogastric cancer patients were interviewed (see chapter 6). Patients described a substantial impact of chemosensory and food-related changes on daily life (by changing daily routines), social life (eating being less sociable) and roles in the household (changing roles in cooking and grocery shopping). Finally, in chapter 7, we assessed the association between self-reported taste and smell perception and quality of life in breast cancer patients. A worse taste and smell perception was associated with a worse global quality of life, role, social and emotional functioning shortly after chemotherapy. In patients treated with trastuzumab, a worse taste and smell perception was still associated with quality of life, social and role functioning half a year after chemotherapy had ended. From the studies in this thesis we can conclude that chemotherapy mainly affects the sense of taste. The subjective perception of taste was associated with a lower preference for food products and lower energy intake. This indicates that it is not necessarily an actual change in the sense of taste or smell that has an impact on patients, but flavour perception as a whole and potentially a lower enjoyment of food. Moreover, these perceived changes in taste and smell can have a substantial impact on cancer patients’ lives, in a practical way by changing daily patterns of eating, but also socially and in roles in the household. A changed chemosensory perception during chemotherapy may lead to a worsened nutritional status, and could thereby negatively impact the response to chemotherapy. Therefore chemosensory perception should be monitored during chemotherapy. Future studies should further investigate the mechanisms behind chemosensory changes, factors that contribute to subjective taste perception and possible interventions to alleviate chemosensory changes during chemotherapy.

Background: Well-trained elite athletes differ from the general population in being considerably more physically active and by other lifestyle characteristics including intensive training routines and periodisation of their training programs. Hence, adequate intake of energy and nutrients is of great importance to this population to ensure optimal performance and recovery during training or competition and also to minimize health risks. A consistent dietary intake pattern, in line with the sport-specific recommendations can be difficult to achieve for this group. The specific recommendations are formulated for nutritional intake during and after training or within competition. However, a large variation is seen in dietary intake by athletes. Therefore, the question arises as to what extent athletes meet recommendations and use nutritional supplements in an optimal manner. Aims: First, to investigate dietary intake and nutritional supplement use by well-trained Dutch athletes and compare these intakes with recommendations both for the general population and sport nutrition recommendations, which are based on expert consensus. Second, to provide an up-to-date overview of nutrient intake levels in a diverse and relatively large group of Dutch elite and sub-elite athletes practicing sports at the highest competitive level. Methods: As part of this thesis 24-hour recalls and questionnaires were used to gain insight into dietary intake and nutritional supplement use (n=553). To validate our methods, 24-hour nitrogen urine excretions were obtained in a subsample of our athletic population (n=47). A questionnaire was used to 1) investigate the prevalence of nutritional supplement use in a large sample of the athletic population (n=778) and 2) investigate the prevalence of nutritional supplement use in a large sample of the Dutch general population (n=1544). Finally, food intake during an ultramarathon was monitored (n=4) and questioned using a food frequency questionnaire (n=41). Results: Our validation study showed that 24-hour recalls and accompanying questionnaires underestimated protein intake in young elite athletes to the same extent as reported for non- athlete populations. Notwithstanding this, the method was considered suitable for ranking athletes according to their protein intake as needed in epidemiological studies. It was found that most athletes were able to meet the estimated average requirement (EAR) for carbohydrate and protein. Regarding sport nutrition recommendations, most of the athletes met protein (1.2 g/kg) but not carbohydrate recommendations (5 g/kg). No major differences in carbohydrate and protein intake were seen between sports categories (i.e. endurance, team and strength athletes). Athletes were at risk of too low intake levels of several micronutrients, especially when they did not use dietary supplements (i.e. vitamin D, vitamin A, vitamin B1 and B2 in men and women, and iron in women), whereas users of supplements showed a slightly elevated risk of intake levels exceeding the upper intake level (UL). This was in particular the case for vitamin B3. Our investigations in ultramarathon runners showed that these athletes did not reach sports nutrition recommendations from their habitual diet. In men and women, habitual mean carbohydrate (CHO) intake was lower than recommended, as was mean protein intake by women. CHO intake during the race was

Background and aim: The worldwide increasing prevalence of overweight and obesity raises concerns for health. There is a clear need for preventive strategies, because current preventative interventions have proven to be unsuccessful in the long term. New strategies may be developed based on targets in the small intestine by activating satiety signals. The thesis aimed to investigate small intestinal targets contributing to food intake regulation. These targets included serotonin, the vagal nerve and the intestinal brake mechanism. Methods: The effects of ileal stimulation with safflower oil (lipid mixture), casein (protein), sucrose (carbohydrate) and rebaudioside A (non-caloric sweetener) on GLP-1 and PYY release were investigated by applying an porcine ex vivo intestinal segment model. The same model was also used to investigate if serotonin is involved in (non-)nutritional-induced GLP-1 and PYY release. The contribution to satiation of GLP-1 and CCK receptors at the vagal nerve, was studied by investigating the effects of GLP-1 and CCK receptor antagonists on ad libitum food intake in a pig model of subdiaphragmatic vagotomy. Two placebo controlled randomized crossover studies were performed in healthy volunteers to investigate the effects of small intestinal macronutrient delivery on ad libitum food intake and satiety signals. The first study compared the effects of duodenal, jejunal and ileal casein delivery on ad libitum food intake and satiety signals. The second study investigated if ileal delivery of all three macronutrients results in activation of satiety signals and reduction in ad libitum food intake. In addition, it was investigated if ileal delivery of native casein is efficiently digested and absorbed and does not result in adverse effects. In both studies the nutrients were delivered to the small intestine by inserting a nasointestinal feeding tube in healthy volunteers. Results: All macronutrients and rebaudioside A stimulated GLP-1 and PYY release from ileal tissue segments. Protein and fat stimulated serotonin release. Inhibiting the reuptake of serotonin resulted in enhanced nutrient induced GLP-1, PYY and CCK release. Serotonin stimulated GLP-1 release from enteroendocrine cells via a serotonin receptor mediated process. Results of the in vivo pig study showed that antagonism of the CCK receptor increased food intake in both vagotomized and sham operated pigs. Blocking the GLP-1 receptor did not affect food intake in both groups. The human studies showed that ileal protein delivery inhibited food intake and activated satiety signals as compared to duodenal or jejunal protein delivery. Also, ileal delivery of small quantities (51.7 kcal) of each macronutrient decreased food intake and activated satiety signals. In addition, it was shown that ileal delivery of native casein resulted in a time and concentration depended increase in plasma concentrations of amino acids and did not result in activation of immune responses nor in gastrointestinal complaints. Conclusions: The data presented in this thesis show that ileal delivery of all macronutrients results in activation of satiety signals and reduction of food intake. Stimulation of the ileum resulted in the strongest activation of satiety signals and inhibition of food intake compared to duodenal and jejunal stimulation. Besides direct nutrient-receptor interaction, the ileum senses (non-)nutritional stimuli via serotonin mediated processes resulting in GLP-1 release. In conclusion, these results demonstrate that targeting the ileum with small amounts of macronutrients is safe and has potential as a weight management strategy.

Measurement errors in dietary assessment using duplicate portions as reference method Laura Trijsburg Background: As Food Frequency Questionnaires (FFQs) are subject to measurement error, associations between self-reported intake by FFQ and outcome measures should be corrected for measurement error with data from a reference method. Whether the correction is adequate depends on the characteristics of the reference method used in the validation study. The duplicate portion method (DP), compared to the often used 24h recall (24hR), seems a promising reference method as correlated errors between FFQ and DP, such as memory bias, errors in portion size estimations and food composition databases, are not expected. Aim: This thesis aimed to determine the validity of the DP compared to the 24hR as a reference method for FFQ validation. The second aim was to explore the validity of nutrient densities for DP, 24hR and FFQ. The third aim was to determine the factors associated with misreporting of energy, protein and potassium as estimated by DP, 24hR and FFQ. Methods: Within the DuPLO-study, a Dutch validation study which is part of the NQplus study, two DPs, two FFQs, two blood and urinary biomarkers and one to fifteen 24hRs (web-based and/or telephone-based) were collected in 198 subjects, within 1.5 years. Also, one or two doubly labelled water measurements were available for 69 participants. Multivariate measurement error models were used to assess proportional scaling bias, error correlations with the FFQ, validity coefficients and attenuation factors. Furthermore linear regression analysis was used to determine the association between misreporting and various factors. Results: The DP was less influenced by proportional scaling bias, had lower correlated errors with the FFQ and showed higher attenuation factors than the 24hR for potassium, sodium and protein. Also, the DP seemed a better reference method than the 24hR for the assessment of validity coefficients for the FFQ for various fatty acids. The attenuation factors for the FFQ, using either the DP or 24hR as reference method, agreed reasonably well. Furthermore, the DP showed, when using plasma fatty acids as reference, slightly better ranking of participants according to their intake of n-3 fatty acids (0.33) and the n‑3/LA ratio (0.34) than the 24hR (0.22 and 0.24, respectively). Less group level bias was observed for protein and sodium densities compared to their absolute intakes for FFQ, 24hR and DP, but not for potassium. Overall the validity coefficients and attenuation factors for DP, 24hR and FFQ did not improve for nutrient densities compared to absolute intakes, except for the attenuation factor for sodium density. Lastly, BMI proved to be the most consistent determinant associated with misreporting (group level bias) of energy, protein and potassium for DP, 24hR and FFQ. Men tended to underreport protein by the DP, FFQ and 24hR and persons of older age underreported potassium but only by the 24hR and FFQ. Other explorative determinants did not show a consistent association with misreporting of energy or nutrients by the different dietary assessment methods. Conclusion: With respect to error correlations and attenuation factors the DP performed slightly better than the 24hR as a reference method for validating FFQs in epidemiological research. Furthermore, the use of nutrient densities does not necessarily improve the validity of the dietary intake estimates from DP, 24hR and FFQ. Moreover, it was shown that BMI is an important determinant of misreporting of energy, protein and potassium for these three assessment methods.

The main, most important function of roots belonging to horticultural crops is the uptake of water and nutrients. Healthy roots are essential for a healthy plant. After all, if the uptake of water and nutrients is not functioning properly, then other aspects also leave a lot to be desired

Measurement errors in dietary assessment using duplicate portions as reference method Laura Trijsburg Background: As Food Frequency Questionnaires (FFQs) are subject to measurement error, associations between self-reported intake by FFQ and outcome measures should be corrected for measurement error with data from a reference method. Whether the correction is adequate depends on the characteristics of the reference method used in the validation study. The duplicate portion method (DP), compared to the often used 24h recall (24hR), seems a promising reference method as correlated errors between FFQ and DP, such as memory bias, errors in portion size estimations and food composition databases, are not expected. Aim: This thesis aimed to determine the validity of the DP compared to the 24hR as a reference method for FFQ validation. The second aim was to explore the validity of nutrient densities for DP, 24hR and FFQ. The third aim was to determine the factors associated with misreporting of energy, protein and potassium as estimated by DP, 24hR and FFQ. Methods: Within the DuPLO-study, a Dutch validation study which is part of the NQplus study, two DPs, two FFQs, two blood and urinary biomarkers and one to fifteen 24hRs (web-based and/or telephone-based) were collected in 198 subjects, within 1.5 years. Also, one or two doubly labelled water measurements were available for 69 participants. Multivariate measurement error models were used to assess proportional scaling bias, error correlations with the FFQ, validity coefficients and attenuation factors. Furthermore linear regression analysis was used to determine the association between misreporting and various factors. Results: The DP was less influenced by proportional scaling bias, had lower correlated errors with the FFQ and showed higher attenuation factors than the 24hR for potassium, sodium and protein. Also, the DP seemed a better reference method than the 24hR for the assessment of validity coefficients for the FFQ for various fatty acids. The attenuation factors for the FFQ, using either the DP or 24hR as reference method, agreed reasonably well. Furthermore, the DP showed, when using plasma fatty acids as reference, slightly better ranking of participants according to their intake of n-3 fatty acids (0.33) and the n‑3/LA ratio (0.34) than the 24hR (0.22 and 0.24, respectively). Less group level bias was observed for protein and sodium densities compared to their absolute intakes for FFQ, 24hR and DP, but not for potassium. Overall the validity coefficients and attenuation factors for DP, 24hR and FFQ did not improve for nutrient densities compared to absolute intakes, except for the attenuation factor for sodium density. Lastly, BMI proved to be the most consistent determinant associated with misreporting (group level bias) of energy, protein and potassium for DP, 24hR and FFQ. Men tended to underreport protein by the DP, FFQ and 24hR and persons of older age underreported potassium but only by the 24hR and FFQ. Other explorative determinants did not show a consistent association with misreporting of energy or nutrients by the different dietary assessment methods. Conclusion: With respect to error correlations and attenuation factors the DP performed slightly better than the 24hR as a reference method for validating FFQs in epidemiological research. Furthermore, the use of nutrient densities does not necessarily improve the validity of the dietary intake estimates from DP, 24hR and FFQ. Moreover, it was shown that BMI is an important determinant of misreporting of energy, protein and potassium for these three assessment methods.

Background and aim: The worldwide increasing prevalence of overweight and obesity raises concerns for health. There is a clear need for preventive strategies, because current preventative interventions have proven to be unsuccessful in the long term. New strategies may be developed based on targets in the small intestine by activating satiety signals. The thesis aimed to investigate small intestinal targets contributing to food intake regulation. These targets included serotonin, the vagal nerve and the intestinal brake mechanism. Methods: The effects of ileal stimulation with safflower oil (lipid mixture), casein (protein), sucrose (carbohydrate) and rebaudioside A (non-caloric sweetener) on GLP-1 and PYY release were investigated by applying an porcine ex vivo intestinal segment model. The same model was also used to investigate if serotonin is involved in (non-)nutritional-induced GLP-1 and PYY release. The contribution to satiation of GLP-1 and CCK receptors at the vagal nerve, was studied by investigating the effects of GLP-1 and CCK receptor antagonists on ad libitum food intake in a pig model of subdiaphragmatic vagotomy. Two placebo controlled randomized crossover studies were performed in healthy volunteers to investigate the effects of small intestinal macronutrient delivery on ad libitum food intake and satiety signals. The first study compared the effects of duodenal, jejunal and ileal casein delivery on ad libitum food intake and satiety signals. The second study investigated if ileal delivery of all three macronutrients results in activation of satiety signals and reduction in ad libitum food intake. In addition, it was investigated if ileal delivery of native casein is efficiently digested and absorbed and does not result in adverse effects. In both studies the nutrients were delivered to the small intestine by inserting a nasointestinal feeding tube in healthy volunteers. Results: All macronutrients and rebaudioside A stimulated GLP-1 and PYY release from ileal tissue segments. Protein and fat stimulated serotonin release. Inhibiting the reuptake of serotonin resulted in enhanced nutrient induced GLP-1, PYY and CCK release. Serotonin stimulated GLP-1 release from enteroendocrine cells via a serotonin receptor mediated process. Results of the in vivo pig study showed that antagonism of the CCK receptor increased food intake in both vagotomized and sham operated pigs. Blocking the GLP-1 receptor did not affect food intake in both groups. The human studies showed that ileal protein delivery inhibited food intake and activated satiety signals as compared to duodenal or jejunal protein delivery. Also, ileal delivery of small quantities (51.7 kcal) of each macronutrient decreased food intake and activated satiety signals. In addition, it was shown that ileal delivery of native casein resulted in a time and concentration depended increase in plasma concentrations of amino acids and did not result in activation of immune responses nor in gastrointestinal complaints. Conclusions: The data presented in this thesis show that ileal delivery of all macronutrients results in activation of satiety signals and reduction of food intake. Stimulation of the ileum resulted in the strongest activation of satiety signals and inhibition of food intake compared to duodenal and jejunal stimulation. Besides direct nutrient-receptor interaction, the ileum senses (non-)nutritional stimuli via serotonin mediated processes resulting in GLP-1 release. In conclusion, these results demonstrate that targeting the ileum with small amounts of macronutrients is safe and has potential as a weight management strategy.

Appetite is often reduced in patients with chronic illness, including cancer. Cancer anorexia, loss of appetite, frequently co-exists with cachexia, and the combined clinical picture is known as anorexia-cachexia syndrome. In patients suffering from this syndrome, anorexia considerably contributes to the progression of cachexia, and strongly impinges on quality of life. Inflammatory processes in the hypothalamus are considered to play a crucial role in the development of disease-related anorexia. The main aim of this thesis was to further elucidate crucial processes involved in the pathogenesis of anorexia in cancer. To investigate mechanisms specifically involved in cancer anorexia, we used two tumour mouse models with opposing food intake behaviours: a C26-colon adenocarcinoma model with increased food intake and a Lewis lung carcinoma model with decreased food intake. In both models, tumour-induced cachexia (body wasting) was strongly present. The contrast in food intake behaviour between tumour-bearing (TB) mice in response to growth of the two different tumours was used to distinguish processes involved in cachexia from those specifically involved in anorexia. The hypothalamus was used for transcriptomic analysis (Affymetrix chips). We found expression of genes involved in serotonin signalling in the hypothalamus to be differentially regulated between the two tumour models. Furthermore, transcriptional activity of genes involved in serotonin signalling were inversely associated with food intake behaviour. Surprisingly, we also found a strong increase in gene expression of NPY and AgRP, potent orexigenic neuropeptides, in both models, meaning that their expression did not reflect food intake behaviour. However, NPY has also been described to regulate energy storage. Therefore, we hypothesized that this upregulation of NPY/AgRP corresponded to weight loss, which was severe in both tumour models. Using hypothalamic cell lines we further explored how serotonin might act on food intake regulatory pathways. We showed that serotonin was able to inhibit neuronal NPY secretion, while not affecting gene expression. Inflammatory markers IL-6 and TNFα were also measured in plasma and it was found that C26 TB mice had a lower inflammatory response than LL TB mice. These differences in inflammatory response could be implicated in the differences in feeding behaviour and serotonin signalling between C26 and LL TB mice. We therefore investigated the direct influence of inflammation on hypothalamic serotonin turnover and its contribution to the development of anorexia. To this end, different doses of TNF and IL-6 were administered by injection to healthy mice, inducing an acute inflammatory response. The injected cytokine doses were estimated from their corresponding plasma levels measured in tumour bearing (TB) mice. Also in this cytokine induced-anorexia model, where anorexia was exclusively induced by an inflammatory response, serotonin metabolism in the hypothalamus was affected. Both TNF and IL-6 increased hypothalamic serotonin turnover while also inducing anorectic behaviour. Furthermore, the effect of cytokines on increasing serotonin turnover was supported by in vitro experiments with hypothalamic neuronal cell lines. In conclusion, we identified hypothalamic serotonin signalling to play a major role in the decrease in food intake during cancer. Serotonin signalling itself is modulated by inflammatory mediators. Therefore, hypothalamic inflammation is an important trigger in the failure of hypothalamic food-intake regulation, probably by affecting serotonergic signalling, which acts as an upstream modulator of various orexigenic and anorexigenic systems.

Background and aim Children’s vegetable intake is far below that recommended. Despite increased awareness of the importance of vegetable consumption for health, it remains challenging to improve children’s vegetable intake. Since food preferences are central to food intake, it is important to understand how they are shaped and which factors play a role in this. So far, research on the formation of vegetable preferences has focused mainly on infants and school age children but is not elaborately investigated in toddlers/pre-schoolers. Therefore the aim of this thesis was to investigate the underlying mechanisms and modifying factors that play a role in developing 2–5-year-old children’s acceptance of vegetables. Effects of different learning mechanisms, strategies, and modifying factors were explored by diverse studies, including four intervention studies in ecological settings (day-care centres and at home). In another study, we compared 10 intervention studies across Europe. Methods We conducted a series of day-care and in-home interventions. Healthy toddlers and pre-school children participated in the studies. Vegetable liking was measured by relative preference, and consumption was measured by (ad libitum) intake. First, we studied the underlying mechanisms – flavour–nutrient learning, flavour–flavour learning, and repeated exposure – involved in the development toddlers’ food preferences in the short and long term. Novel products like green vegetable soups and vegetable crisps were used as test products, using within-subject designs. The soups differed in energy density to test flavour–nutrient learning (n=28), and the crisps were offered with different dips to test flavour–flavour learning (n=39). Next, we investigated the efficacy of other strategies like taste modification (n=103) and choice-offering (n=70) on 2–5-year-old children’s vegetable liking and intake, using between-subjects designs. Children consumed different vegetable products at home at dinnertime and therefore we used more familiar vegetables as test products. Finally, we combined the results of 10 intervention studies across Europe to explore the influence of individual child characteristics such as breastfeeding history and breastfeeding duration, age, gender, and food neophobia on 2– 6-year-old children’s (n=750) actual vegetable intake. Results We found a clear and persistent effect of repeatedly offering novel and/or disliked vegetables on 2–5-year-old children’s intake. Results for preferences were inconsistent across the studies. We found no strong evidence that strategies such as flavour–flavour learning, flavour–nutrient learning, diluting/hiding a vegetable were more effective in changing vegetable preference than repeated exposure alone. We observed a small positive effect of choice-offering; this strategy could possibly be effective in somewhat older children who already like vegetables, to increase their consumption volume. Factors like breastfeeding duration, vegetable liking, and food neophobia were important for children’s vegetable intake. Children who were more reluctant to try novel food had lower vegetable intake and were not responsive to strategies like repeated exposure, blending, mixing, or hiding vegetables. Longer breastfeeding duration was positively associated with a higher vegetable intake by 2–6-year-old children across three European countries. Gender and age had no influence. Conclusions This thesis demonstrates that repeatedly offering a novel or disliked vegetable in a trusted positive environment is highly effective in promoting toddlers’ and pre-school children’s vegetable intake. Repeated exposure seems to be the way to teach young children to accept novel or disliked foods. Other strategies such as flavouring, adding energy, or taste modification may be helpful in promoting young children’s willingness to try and taste vegetables. Additional strategies such as choice-offering are needed to promote intake of already liked/familiar vegetables when children get older. Individual differences in child characteristics such as food neophobia, breastfeeding duration, and age play a role in shaping food preferences and therefore should get more attention in strategies to promote children’s vegetable acceptance. These results can be used by parents, caregivers, and public health organizations to stimulate children’s vegetable consumption to maintain a more balanced diet.

Background and aim Most children do not eat the recommended amount of vegetables. Their low vegetable intake may be attributed to their low preference for vegetables. Since the first years of life is a sensitive period in the development of taste preferences, and since taste preferences track over time, we started a longitudinal intervention study to investigate the effect of starting weaning with repeated exposure to vegetable purées on vegetable intake and liking during the first 2 years of life. The primary aim of this thesis was to investigate whether starting weaning with vegetables compared to weaning with fruit had an effect on the intake and liking of vegetables on the short and on the long term. The secondary aim was to investigate whether starting weaning with vegetables compared to weaning with fruit, influenced the preferences for sweet taste and daily intake of sugar. Methods First, we conducted an intervention study (n = 101) that investigated the effects of repeated exposure to either vegetable purées (vegetable groups) or fruit purées (fruit groups) on infants’ acceptance of vegetable or fruit purées during the first 18 days of weaning. Intake of the purées and mothers’ rated liking were measured in the lab. From a subsample (n = 60), we also measured liking by analysing the infants’ facial expressions and behaviour after consuming green beans purée in the lab. In two follow-up studies, when the infants were 12 (n = 84) and 23 months of age (n = 81), the long-term effect of the intervention was measured on intake and mothers’ rated liking of the purées in the lab. Additionally, infants’ daily vegetable intake was assessed with 3-day food records at both follow-ups. At the second follow-up, also the influence of starting with vegetables or fruits on children’s preferences for sweet and salty tastes (n = 81), were measured with sweetened and salted water solution and by calculating their daily mono- and disaccharides intake from the 3-day food records. Additionally, a systematic review investigated the current status of knowledge about effective strategies to increase vegetable intake in children younger than 3 y. Results The studies showed that the group of children who were repeatedly exposed to vegetables increased their vegetable intake from 24 ± 28 g to 45 ± 44g (p < 0.001), while the children who were repeatedly exposed to fruit increased their fruit intake from 46 ± 40 g to 66 ± 42 g (p < 0.05) . Interestingly, the first vegetable intake in the fruit group, which was directly after the 18 days of exposure to fruit purées, was as low as the first vegetable intake of the children in the vegetable group at day 1. This indicates that the repeated exposure to fruit did not influence the children’s vegetable intake. These results were confirmed by the results of the facial expressions, showing a decrease in negative facial expressions after repeated exposure to green beans. At the follow-ups, when the infants were 12 and 23 months of age, no differences between the vegetable and fruit groups in green beans or apple purée were found in the lab. Daily intake of vegetables at 12 months of age, was 38% higher (p = 0.02) in the vegetable group (75 ± 43 g) than in the fruit group (54 ± 29 g). At 23 months of age, no significant difference in daily vegetable intake was found between the groups. Also the 23 month olds’ preference for sweet water solutions and their daily mono- and disaccharides intake did not differ between groups. Finally, the systematic review of literature showed that counselling of the parents on healthy eating and nutrition did have a positive long-term effect on their children’s’ vegetable intake, although the effect was relatively small. The review further showed that repeated exposure was the most studied and also the most effective strategy, since all studies reported an increase in intake after repeated exposure to a vegetable. Also exposure to a variety of vegetables showed to have a positive effect on the intake of a new vegetable. Conclusion Weaning with repeated exposure to vegetables has a positive influence on vegetable intake until at least 12 months of age.

Background and aim Most children do not eat the recommended amount of vegetables. Their low vegetable intake may be attributed to their low preference for vegetables. Since the first years of life is a sensitive period in the development of taste preferences, and since taste preferences track over time, we started a longitudinal intervention study to investigate the effect of starting weaning with repeated exposure to vegetable purées on vegetable intake and liking during the first 2 years of life. The primary aim of this thesis was to investigate whether starting weaning with vegetables compared to weaning with fruit had an effect on the intake and liking of vegetables on the short and on the long term. The secondary aim was to investigate whether starting weaning with vegetables compared to weaning with fruit, influenced the preferences for sweet taste and daily intake of sugar. Methods First, we conducted an intervention study (n = 101) that investigated the effects of repeated exposure to either vegetable purées (vegetable groups) or fruit purées (fruit groups) on infants’ acceptance of vegetable or fruit purées during the first 18 days of weaning. Intake of the purées and mothers’ rated liking were measured in the lab. From a subsample (n = 60), we also measured liking by analysing the infants’ facial expressions and behaviour after consuming green beans purée in the lab. In two follow-up studies, when the infants were 12 (n = 84) and 23 months of age (n = 81), the long-term effect of the intervention was measured on intake and mothers’ rated liking of the purées in the lab. Additionally, infants’ daily vegetable intake was assessed with 3-day food records at both follow-ups. At the second follow-up, also the influence of starting with vegetables or fruits on children’s preferences for sweet and salty tastes (n = 81), were measured with sweetened and salted water solution and by calculating their daily mono- and disaccharides intake from the 3-day food records. Additionally, a systematic review investigated the current status of knowledge about effective strategies to increase vegetable intake in children younger than 3 y. Results The studies showed that the group of children who were repeatedly exposed to vegetables increased their vegetable intake from 24 ± 28 g to 45 ± 44g (p < 0.001), while the children who were repeatedly exposed to fruit increased their fruit intake from 46 ± 40 g to 66 ± 42 g (p < 0.05) . Interestingly, the first vegetable intake in the fruit group, which was directly after the 18 days of exposure to fruit purées, was as low as the first vegetable intake of the children in the vegetable group at day 1. This indicates that the repeated exposure to fruit did not influence the children’s vegetable intake. These results were confirmed by the results of the facial expressions, showing a decrease in negative facial expressions after repeated exposure to green beans. At the follow-ups, when the infants were 12 and 23 months of age, no differences between the vegetable and fruit groups in green beans or apple purée were found in the lab. Daily intake of vegetables at 12 months of age, was 38% higher (p = 0.02) in the vegetable group (75 ± 43 g) than in the fruit group (54 ± 29 g). At 23 months of age, no significant difference in daily vegetable intake was found between the groups. Also the 23 month olds’ preference for sweet water solutions and their daily mono- and disaccharides intake did not differ between groups. Finally, the systematic review of literature showed that counselling of the parents on healthy eating and nutrition did have a positive long-term effect on their children’s’ vegetable intake, although the effect was relatively small. The review further showed that repeated exposure was the most studied and also the most effective strategy, since all studies reported an increase in intake after repeated exposure to a vegetable. Also exposure to a variety of vegetables showed to have a positive effect on the intake of a new vegetable. Conclusion Weaning with repeated exposure to vegetables has a positive influence on vegetable intake until at least 12 months of age.

Background and aim Children’s vegetable intake is far below that recommended. Despite increased awareness of the importance of vegetable consumption for health, it remains challenging to improve children’s vegetable intake. Since food preferences are central to food intake, it is important to understand how they are shaped and which factors play a role in this. So far, research on the formation of vegetable preferences has focused mainly on infants and school age children but is not elaborately investigated in toddlers/pre-schoolers. Therefore the aim of this thesis was to investigate the underlying mechanisms and modifying factors that play a role in developing 2–5-year-old children’s acceptance of vegetables. Effects of different learning mechanisms, strategies, and modifying factors were explored by diverse studies, including four intervention studies in ecological settings (day-care centres and at home). In another study, we compared 10 intervention studies across Europe. Methods We conducted a series of day-care and in-home interventions. Healthy toddlers and pre-school children participated in the studies. Vegetable liking was measured by relative preference, and consumption was measured by (ad libitum) intake. First, we studied the underlying mechanisms – flavour–nutrient learning, flavour–flavour learning, and repeated exposure – involved in the development toddlers’ food preferences in the short and long term. Novel products like green vegetable soups and vegetable crisps were used as test products, using within-subject designs. The soups differed in energy density to test flavour–nutrient learning (n=28), and the crisps were offered with different dips to test flavour–flavour learning (n=39). Next, we investigated the efficacy of other strategies like taste modification (n=103) and choice-offering (n=70) on 2–5-year-old children’s vegetable liking and intake, using between-subjects designs. Children consumed different vegetable products at home at dinnertime and therefore we used more familiar vegetables as test products. Finally, we combined the results of 10 intervention studies across Europe to explore the influence of individual child characteristics such as breastfeeding history and breastfeeding duration, age, gender, and food neophobia on 2– 6-year-old children’s (n=750) actual vegetable intake. Results We found a clear and persistent effect of repeatedly offering novel and/or disliked vegetables on 2–5-year-old children’s intake. Results for preferences were inconsistent across the studies. We found no strong evidence that strategies such as flavour–flavour learning, flavour–nutrient learning, diluting/hiding a vegetable were more effective in changing vegetable preference than repeated exposure alone. We observed a small positive effect of choice-offering; this strategy could possibly be effective in somewhat older children who already like vegetables, to increase their consumption volume. Factors like breastfeeding duration, vegetable liking, and food neophobia were important for children’s vegetable intake. Children who were more reluctant to try novel food had lower vegetable intake and were not responsive to strategies like repeated exposure, blending, mixing, or hiding vegetables. Longer breastfeeding duration was positively associated with a higher vegetable intake by 2–6-year-old children across three European countries. Gender and age had no influence. Conclusions This thesis demonstrates that repeatedly offering a novel or disliked vegetable in a trusted positive environment is highly effective in promoting toddlers’ and pre-school children’s vegetable intake. Repeated exposure seems to be the way to teach young children to accept novel or disliked foods. Other strategies such as flavouring, adding energy, or taste modification may be helpful in promoting young children’s willingness to try and taste vegetables. Additional strategies such as choice-offering are needed to promote intake of already liked/familiar vegetables when children get older. Individual differences in child characteristics such as food neophobia, breastfeeding duration, and age play a role in shaping food preferences and therefore should get more attention in strategies to promote children’s vegetable acceptance. These results can be used by parents, caregivers, and public health organizations to stimulate children’s vegetable consumption to maintain a more balanced diet.

Appetite is often reduced in patients with chronic illness, including cancer. Cancer anorexia, loss of appetite, frequently co-exists with cachexia, and the combined clinical picture is known as anorexia-cachexia syndrome. In patients suffering from this syndrome, anorexia considerably contributes to the progression of cachexia, and strongly impinges on quality of life. Inflammatory processes in the hypothalamus are considered to play a crucial role in the development of disease-related anorexia. The main aim of this thesis was to further elucidate crucial processes involved in the pathogenesis of anorexia in cancer. To investigate mechanisms specifically involved in cancer anorexia, we used two tumour mouse models with opposing food intake behaviours: a C26-colon adenocarcinoma model with increased food intake and a Lewis lung carcinoma model with decreased food intake. In both models, tumour-induced cachexia (body wasting) was strongly present. The contrast in food intake behaviour between tumour-bearing (TB) mice in response to growth of the two different tumours was used to distinguish processes involved in cachexia from those specifically involved in anorexia. The hypothalamus was used for transcriptomic analysis (Affymetrix chips). We found expression of genes involved in serotonin signalling in the hypothalamus to be differentially regulated between the two tumour models. Furthermore, transcriptional activity of genes involved in serotonin signalling were inversely associated with food intake behaviour. Surprisingly, we also found a strong increase in gene expression of NPY and AgRP, potent orexigenic neuropeptides, in both models, meaning that their expression did not reflect food intake behaviour. However, NPY has also been described to regulate energy storage. Therefore, we hypothesized that this upregulation of NPY/AgRP corresponded to weight loss, which was severe in both tumour models. Using hypothalamic cell lines we further explored how serotonin might act on food intake regulatory pathways. We showed that serotonin was able to inhibit neuronal NPY secretion, while not affecting gene expression. Inflammatory markers IL-6 and TNFα were also measured in plasma and it was found that C26 TB mice had a lower inflammatory response than LL TB mice. These differences in inflammatory response could be implicated in the differences in feeding behaviour and serotonin signalling between C26 and LL TB mice. We therefore investigated the direct influence of inflammation on hypothalamic serotonin turnover and its contribution to the development of anorexia. To this end, different doses of TNF and IL-6 were administered by injection to healthy mice, inducing an acute inflammatory response. The injected cytokine doses were estimated from their corresponding plasma levels measured in tumour bearing (TB) mice. Also in this cytokine induced-anorexia model, where anorexia was exclusively induced by an inflammatory response, serotonin metabolism in the hypothalamus was affected. Both TNF and IL-6 increased hypothalamic serotonin turnover while also inducing anorectic behaviour. Furthermore, the effect of cytokines on increasing serotonin turnover was supported by in vitro experiments with hypothalamic neuronal cell lines. In conclusion, we identified hypothalamic serotonin signalling to play a major role in the decrease in food intake during cancer. Serotonin signalling itself is modulated by inflammatory mediators. Therefore, hypothalamic inflammation is an important trigger in the failure of hypothalamic food-intake regulation, probably by affecting serotonergic signalling, which acts as an upstream modulator of various orexigenic and anorexigenic systems.

Doel van dit onderzoek was: meten van de reproduceerbaarheid van een bestaande voedselfrequentievragenlijst (VVL) bij een groep patiënten met ongewijzigd dieetadvies; meten van verandering in energie-inname bij een groep patiënten met gewijzigde dieetadvies; en evalueren van de bruikbaarheid van VVL bij patiënt en diëtist

The main, most important function of roots belonging to horticultural crops is the uptake of water and nutrients. Healthy roots are essential for a healthy plant. After all, if the uptake of water and nutrients is not functioning properly, then other aspects also leave a lot to be desired

For many people, food intake management is a challenging process, as food is always in abundance and the appetite control system is challenged and potentially overpowered by habits, routines and cues in the external environment. The present thesis focuses on satiation and satiety expectations and inferences as a guide for food intake, both within and across consumption episodes. More specifically, the role of physiological cues, claims on food packages and packaging design in the development of satiation/ satiety expectations and their effect on food intake is studied. This thesis takes as a starting point that feedback from previous consumption experiences is important for the development of satiety and satiation expectations. In addition, it is expected that satiation and satiety expectations also can be inferred ‘on the spot’, either explicitly (as from satiety claims), but potential also implicitly and more intuitively (as from packaging design and other factors in the eating context).

For many people, food intake management is a challenging process, as food is always in abundance and the appetite control system is challenged and potentially overpowered by habits, routines and cues in the external environment. The present thesis focuses on satiation and satiety expectations and inferences as a guide for food intake, both within and across consumption episodes. More specifically, the role of physiological cues, claims on food packages and packaging design in the development of satiation/ satiety expectations and their effect on food intake is studied. This thesis takes as a starting point that feedback from previous consumption experiences is important for the development of satiety and satiation expectations. In addition, it is expected that satiation and satiety expectations also can be inferred ‘on the spot’, either explicitly (as from satiety claims), but potential also implicitly and more intuitively (as from packaging design and other factors in the eating context).

Consumers eat at various sequential occasions throughout the day. The current thesis addresses the question of how one consumption episode can affect the amount of consumption at a subsequent episode. The thesis focuses specifically on how the social context during a consumption episode affects subsequent consumption, and on when consumers rely on hunger and satiety cues in sequential consumption episodes. Dit proefschrift richt zich op de vraag hoe een consumptiemoment de hoeveelheid consumptie op een later moment kan beïnvloeden. Gedurende de dag eten consumenten op verschillende momenten. Specifiek richt dit proefschrift zich op de vraag hoe de sociale context van een consumptie-episode latere consumptie beïnvloedt en op de vraag wanneer consumenten zich laten leiden door honger en verzadigingssignalen in opeenvolgende consumptiemomenten.

Consumers eat at various sequential occasions throughout the day. The current thesis addresses the question of how one consumption episode can affect the amount of consumption at a subsequent episode. The thesis focuses specifically on how the social context during a consumption episode affects subsequent consumption, and on when consumers rely on hunger and satiety cues in sequential consumption episodes.

Vrouwelijke en mannelijke Wistar ratten kregen via het drinkwater patuline toegediend in concentraties van 0, 24, 84 of 295 mg/l citraat buffer (1mM) gedurende 4 weken. In deze studie zijn de resultaten van een recente subacute toxiciteitsstudie met toediening van patuline via het drinkwater bevestigd. Vergeleken met de controlegroep is voedsel- en vochtopname in het midden- en hoogste doseringsgroep en lichaamsgewicht in de hoogste doseringsgroep afgenomen. De kratinineklaringin de hoogste doseringsgroep was verlaagd. Morfologisch gezien kon geen glomerulaire schade worden aangetoond. In de hoogste doseringsgroep zijn ulcera in de maagfundus en vergroting en activatie van de pancreatico-duodenale lymfklieren aangetoond, terwijl hyperaemie van de villi in het duodenum werd waargenomen in de midden en hoogste doseringsgroep.

The increases in obesity prevalence coincide with changes in our food environment, such as an increased consumption of processed, energy dense foods. This suggests that the foods we consume are at least partly responsible for the obesity epidemic. The aim of this thesis is therefore to investigate food characteristics, with the focus on taste, eating rate and energy density, and their relation to dietary intake. Taste is studied in two respects. First, the contribution of taste qualities to the diet is investigated, using the Food Consumption Survey 2003. Foods are classified according to their predominant taste (sweet, salty or savoury, sour, bitter or neutral). Energy intake of the foods within taste categories is assessed, showing that the largest part (34%) of the daily energy intake originates from sweet foods. Second, it is investigated whether taste, which is supposed to be a nutrient sensor, can fulfil this function within the current diet. Intensities of the five basic tastes of 50 commonly consumed foods are therefore assessed and associated with the nutrient content. Positive associations are found between sweetness and mono- & disaccharides and between both saltiness and savouriness and sodium and protein. The associations are less pronounced in highly processed foods, which suggests that in these foods the ability to sense nutrient content based on taste is limited. The influences of an incongruence between sensory properties and nutrient content are also investigated, byexamining the effects of fat perceptionon energy intake. We demonstrated that energy intake is almost 10% lower in case of visible fats compared to hidden fats, suggesting that hidden fats may contribute to overconsumption. Eating rateseems to be associated with food intake. The contribution of eating rate to the diet is investigated, using the Food Consumption Survey 2003. Foods are classified into one of four eating rate categories, and energy intake of the foods within each category is assessed. Results demonstrate that foods with slow calories (kJ/min) provide 10%, whereas foods with fast calories provide 37% to the daily energy intake. So in the current diet, the consumption of foods with a high eating rate is high. The effects of eating rate on intake are also investigated, showing that eatingrate is positively associated with food and energy intake. People may therefore be at risk of overconsumption, when consuming foods with a high eating rate. Consuming energy dense snacks is often blamed for affecting energy balance, but findings are inconclusive. Therefore, effects of snack consumption on body weight are investigated. No changes in body weight are observed after 8 weeks, when energy density of snacks was either low or high. This suggests that consuming snacks does not necessarily contribute to weight gain, at least in normal-weight young adults. In conclusion,when taste and other sensory properties do not accurately reflect the nutrient content, which applies particularly to highly processed foods, this may lead to high food intakes. In addition, a large part of the daily energy intake originates from foods with a high eating rate, which stimulates food and energy intake. So the high eating rate of the foods in the current diet may be responsible for overconsumption. These findings may be helpful in following the recommendations of the Nutrition Centre to lose weight. Last, even though we did not find evidence that consuming energy dense snacks results in weight gain, the advice should nevertheless be to limit the intake of energy dense foods, at least until evidence becomes more conclusive.

Introduction Food texture has been shown to be an important factor in the regulation of food (energy) intake. Liquid foods e.g. elicit weaker satiety responses than solid foods with a similar energy content, and texture affects satiation, i.e. ad libitum food intake. Whether theeffect of food texture on food intake stays the same over repeated exposure requires further investigation. Aim The aim of this thesis is to investigate the role of food texture in learned satiation. We assessed the effect of texture on changes in ad libitum intake and expected satiation after repeated consumption of foods with different energy density. Methods We conducted a series of learning experiments with healthy young adults. Participants repeatedly consumed a low-energy-dense (LED) and a high-energy-dense (HED) yogurt, which were either low (n=24) or high (n=22) in viscosity in one study; and consumed with a straw (liquid yogurt, n=34) or with a spoon (liquid yogurt, n=36; semi-solid yogurt, n=35) in a second study. Next, we investigated changes in expected satiation and intake after repeated consumption of a LED soup (n=32) or a HED soup (n=32) with similar appearance; and of a liquid and a semi-solid custard with a similar energy density (n=53). Additionally, we assessed the effect of texture, flavour and means of consumption on expected satiation of iso-caloric dairy products in 3 single-meal experiments. Finally, we served a fixed amount of a LED or HED food - either liquid or semi-solid - at each meal on 3 consecutive days, and measured ad libitum buffet intake directly after consumption of these foods (n=27). Results Texture clearly affected satiation:ad libitum intake was up to 30% higher of liquid foods when compared with semi-solids foods in all experiments (p

Background and aim The rise in obesity over the last decades is considered to be related to changes in the food environment. Our current diet exists of foods that facilitate fast intake of energy and minimal oral processing. Various studies showed that higher eating rate leads to higher food intake, and therefore promote energy overconsumption. When consuming at a high eating rate, the food spends less time in the oral cavity, resulting in less sensory exposure per gram food. The exposure to the taste of the food in the oral cavity is potentially important in controlling food intake. The studies in this thesis investigated the principle mechanisms through which orosensory exposure affects satiation. The factors that were studied were taste intensity, oral residence duration and bite size. The impact of these factors and their relative contributions to satiation will provide tools for designing new foods to prevent overconsumption. Methods We conducted five studies. The subjects that participated in the studies were healthy young normal weight adults. Satiation was measured by ad libitum intake and subjective ratings of hunger and fullness. Tomato soup was used as test product in all studies. We started by investigating the effect of taste intensity on ad libitum intake (n=48). Salt was used to vary the taste intensity in soup. We selected two salt concentrations for low-salt and high-salt soup that were similar in pleasantness on an individual basis. In the next study, salt taste intensity in soup was investigated again, but this time we changed the state of hunger (a preload was offered) and the meal composition (subjects were served a second course after the soup) (n=43). In the third study, the impact of taste intensity versus the duration of orosensory exposure (manipulated by changing the bite size) on satiation was investigated, by using peristaltic pumps to control the bites (n=55). The fourth study focussed on the underlying mechanisms of bite size on food intake (n=56). Therefore, separate effects of oral residence duration per gram food and number of bites per gram food on ad libitum intake were assessed. Finally, we investigated if bite size affects the perceived food intake. Subjects estimated the amount consumed after intake with small or large bites, in both focussed and a distracted states (n=53). In addition, effects of distraction on bite size were investigated. Results Taste intensity did not affect ad libitum intake when the soup was presented as single lunch-item in a hungry state. However, higher taste intensity reduced ad libitum intake by ~8%, when the soup was presented after a preload or as a starter followed by a second meal. Smaller bite sizes decreased ad libitum intake by ~25% and did not interact with taste intensity. That smaller bites are more satiating than larger bites was confirmed by hunger and fullness ratings. Hunger decreased faster per consumed gram food when consuming with small bites compared to large bites. A similar effect was found for the increase in fullness. Ad libitum intake was separately reduced by longer oral residence duration and higher number of bites per gram food, there was no interaction between the two variables. Time-intensity measurements showed that both higher number of bites and longer oral residence duration increase the total magnitude of orosensory exposure to the taste of the food. Consumption with large bites resulted in underestimations of the amount consumed, whereas consumption with small bites did not. Distraction increased ad libitum intake. Distraction led to a higher number of bites over the meal but did not affect bite size. Conclusions This thesis demonstrates that consuming foods with smaller bite sizes, longer oral residence durations and higher taste intensities lowers food intake. These effects are possibly explained through their enhancement of the orosensory exposure to the taste of the foods. Consumption with large bites leads to underestimation of the amount that is consumed. An underestimation of the amount consumed is a risk factor for overconsumption. These results could be used by the food industry to enhance the satiating capacity of foods in order to prevent overconsumption and decrease the prevalence of obesity.

As part of a collaboration between the research programme Knowledge for Primary Processes Silt of Rijkswaterstaat Waterdienst NWOB (department of Infrastructure and Environment, MinIenM, RWS) and the Monitoring programme Sand extraction RWS and the LaMER Foundation, RWS-WD NWOB requested further research into the relation between food availability and Ensis production. The aim is to better understand the effect of different algae and silt concentrations on filtration and growth rates and improve prediction of effects. Laboratory experiments were carried out with Ensis directus to estimate food intake rate and growth rate as a function of food density and clam size. Growth experiments carried out in 2010 showed that the species seems to be very fragile as shown by the low growth rates and high mortality rates. Improvements designed to optimize the experimental conditions, survival rates and experimental set-up were implemented in 2011. These were: experimental animals were collected with a box corer instead of a suction dredge; animals were kept in cylinders without sediment, but their shells were closed with elastic bands during the filtration experiments; circular tanks were used with increased water movement; the diet during the growth experiment consisted of two species of algae. Two food levels were tested: low food availability (6.5 μg Chla/l) and high food availability (16.5 μg Chla/l) at four silt concentrations (0, 50, 150 and 300 mg/l). Only the highest silt concentration induced a reduction in filtration rate. Food level did not influence filtration rate of Ensis, but intake rate is higher at the high food concentration, because more algal cells are present in a certain volume of water. Longterm (10 weeks) exposure to silt concentrations of 300 mg/l showed significantly higher growth than the 150 mg/l treatment indicating that exposure to a high silt concentration did not induce a reduction in growth. Long-term (10 weeks) exposure to a food level of 6.5 ug chla per liter reduced shell growth of Ensis compared to growth at 16.5 ug chla per liter. The filtration and growth rate results are used in a modelling study on growth and condition of Ensis during sand extraction 2013-2017 (Schellekens, in prep). The conclusions of this study give more notion of the effects of sand extraction in the coastal zone of the North Sea on the viability of the razor clam Ensis directus. Sand extraction always goes together with an increase of silt concentration in the water column. This reduces the light conditions for algal growth which reduces the food availability for Ensis. The laboratory experiments suggest that Ensis is more sensitive to a reduction in algal concentration than to an increase in silt concentration. Some discussion is given on the implications of the results for the management of sand extraction.

Introduction Food texture has been shown to be an important factor in the regulation of food (energy) intake. Liquid foods e.g. elicit weaker satiety responses than solid foods with a similar energy content, and texture affects satiation, i.e. ad libitum food intake. Whether theeffect of food texture on food intake stays the same over repeated exposure requires further investigation. Aim The aim of this thesis is to investigate the role of food texture in learned satiation. We assessed the effect of texture on changes in ad libitum intake and expected satiation after repeated consumption of foods with different energy density. Methods We conducted a series of learning experiments with healthy young adults. Participants repeatedly consumed a low-energy-dense (LED) and a high-energy-dense (HED) yogurt, which were either low (n=24) or high (n=22) in viscosity in one study; and consumed with a straw (liquid yogurt, n=34) or with a spoon (liquid yogurt, n=36; semi-solid yogurt, n=35) in a second study. Next, we investigated changes in expected satiation and intake after repeated consumption of a LED soup (n=32) or a HED soup (n=32) with similar appearance; and of a liquid and a semi-solid custard with a similar energy density (n=53). Additionally, we assessed the effect of texture, flavour and means of consumption on expected satiation of iso-caloric dairy products in 3 single-meal experiments. Finally, we served a fixed amount of a LED or HED food - either liquid or semi-solid - at each meal on 3 consecutive days, and measured ad libitum buffet intake directly after consumption of these foods (n=27). Results Texture clearly affected satiation:ad libitum intake was up to 30% higher of liquid foods when compared with semi-solids foods in all experiments (p

As part of a collaboration between the research programme Knowledge for Primary Processes Silt of Rijkswaterstaat Waterdienst NWOB (department of Infrastructure and Environment, MinIenM, RWS) and the Monitoring programme Sand extraction RWS and the LaMER Foundation, RWS-WD NWOB requested further research into the relation between food availability and Ensis production. The aim is to better understand the effect of different algae and silt concentrations on filtration and growth rates and improve prediction of effects. Laboratory experiments were carried out with Ensis directus to estimate food intake rate and growth rate as a function of food density and clam size. Growth experiments carried out in 2010 showed that the species seems to be very fragile as shown by the low growth rates and high mortality rates. Improvements designed to optimize the experimental conditions, survival rates and experimental set-up were implemented in 2011. These were: experimental animals were collected with a box corer instead of a suction dredge; animals were kept in cylinders without sediment, but their shells were closed with elastic bands during the filtration experiments; circular tanks were used with increased water movement; the diet during the growth experiment consisted of two species of algae. Two food levels were tested: low food availability (6.5 μg Chla/l) and high food availability (16.5 μg Chla/l) at four silt concentrations (0, 50, 150 and 300 mg/l). Only the highest silt concentration induced a reduction in filtration rate. Food level did not influence filtration rate of Ensis, but intake rate is higher at the high food concentration, because more algal cells are present in a certain volume of water. Longterm (10 weeks) exposure to silt concentrations of 300 mg/l showed significantly higher growth than the 150 mg/l treatment indicating that exposure to a high silt concentration did not induce a reduction in growth. Long-term (10 weeks) exposure to a food level of 6.5 ug chla per liter reduced shell growth of Ensis compared to growth at 16.5 ug chla per liter. The filtration and growth rate results are used in a modelling study on growth and condition of Ensis during sand extraction 2013-2017 (Schellekens, in prep). The conclusions of this study give more notion of the effects of sand extraction in the coastal zone of the North Sea on the viability of the razor clam Ensis directus. Sand extraction always goes together with an increase of silt concentration in the water column. This reduces the light conditions for algal growth which reduces the food availability for Ensis. The laboratory experiments suggest that Ensis is more sensitive to a reduction in algal concentration than to an increase in silt concentration. Some discussion is given on the implications of the results for the management of sand extraction.

The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

Background and aim The sensory attributes of food play a key role in the selection and termination of meals and their rewarding properties. The majority of our foods are either sweet or savoury tasting. In addition, within our food range, savoury-tasting foods contain in general higher levels of protein. The effect of specific taste modalities on human food intake, however, requires further clarification. The primary aim of this thesis was to investigate the role of sweet and savoury taste in food intake and food preferences. The secondary aim was to provide more insight into the processes of explicit and implicit liking and wanting, to be able to identify underlying reward mechanisms involved in food intake behaviour. Methods We conducted series of experiments where healthy young adults participated. We started by investigating the difference between a sweet and savoury taste on satiation, independent of palatability, texture, energy density, and macronutrient composition (n=64). Next, the effect of sweet and savoury taste of a single meal on subsequent satiety and food preferences was investigated (n=61). To further explore the effect of taste in the context of a complete diet on satiety and food preferences, the effect of three 24-h diets that differed only in taste (predominantly sweet tasting, predominantly savoury tasting, or a mixture of sweet and savoury tasting) were compared (n=39). Next, we separated the influence of taste from within-meal protein content on satiety and food preferences, by comparing the effect of sweet and savoury high and low protein single meals (n=60). Finally, the effect of long-term protein status on satiety and food preferences was investigated by comparing the effect of two 14-d diets that differed in protein content (a low protein diet vs. a high protein diet) (n=37). Results Sweet and savoury taste, independent of palatability, texture, energy density, and macronutrient composition, did not differ in their effect on satiation and satiety in terms of subsequent ad libitum intake. Sweet and savoury taste did differ in their effect on subsequent food preferences. In general, after eating a food with a certain taste, appetite for foods with a similar taste was lower than for foods with a dissimilar taste, hence, a clear transfer effect of sensory specific satiety was demonstrated. This transfer effect was not equipotent for sweet and savoury taste; after eating a sweet single meal or sweet 24-h diet, preferences for sweet and savoury foods did not differ. Eating a savoury single meal or savoury 24-h diet, however, led to a clear preference for sweet foods. Neither sweet or savoury tasting single meals nor sweet or savoury 24-h diets shifted food preferences towards high or low protein foods. It was shown that protein content of a meal, independent of taste, did not have an effect on satiety and food preference. We did observe, however, an effect of protein status: after a 14-d low protein diet, there was an increase in ad libitum protein intake, compared to after a 14-d high protein diet, while total energy intake was not different. In addition, food preference for savoury high protein foods was increased. Regarding the different components of food reward it was demonstrated that in all studies both explicit and implicit measures correlated with several aspects of eating. It appeared that in a controlled setting, i.e. in the sensory booths, explicit processes played a stronger determining role in satiation (meal size) than implicit processes. Food choices appeared to be made on a more unconscious level. In a setting where subjects could behave more naturally (i.e. self-selection and serving of foods in a relaxed environment where subjects could sit and eat together), implicit, unconscious processes seemed to explain food intake behaviour more than explicit processes. When subjects experienced protein shortage, after the 14-d low protein diet, it appeared that implicit processes of wanting played a stronger determining role in decisions about what to eat. Conclusion Sweet and savoury taste do not differ in their effect on satiation or satiety in terms of subsequent ad libitum intake. The taste of a meal or diet does have a large effect on subsequent food preferences, thereby showing a clear transfer effect which is not equipotent for sweet and savoury taste. Savoury taste exerts a stronger modulating effect on subsequent food preferences than sweet taste. Sweet and savoury taste of a single meal or 24-h diet do not differ in their effect on food preferences for high or low protein foods. In addition, within-meal protein content seems not to influence satiety or food preferences. A low protein status, however, through selective reduction of dietary protein intake, elicits compensatory changes in food intake and food preferences to restore adequate protein status. It appears that both conscious (explicit) and unconscious (implicit) processes are involved in satiation and food choice. The role implicit motivational processes play in driving food choice is not static, but appears to vary. This is especially the case when homeostasis is challenged (by depleting macronutrient stores), where implicit processes of wanting appear to play a stronger determining role in decisions about what to eat.

In 2009 a survey was conducted among a representative sample of around 500 young people between the ages of 12 and 18 in the Netherlands. The first section examined the eating patterns of the young people and their attitude towards nutrition. The second section examined their vegetable consumption and how they experience vegetables, as well as the factors which influence this. The final section consisted of a choosing experiment, in which the participants were asked to choose from among two or three vegetable products and evaluate them on the basis of a number of product characteristics. In 2009 is een enquêteonderzoek uitgevoerd onder een representatieve steekproefgroep van circa 500 jongeren tussen 12 en 18 jaar in Nederland. In het eerste onderdeel is het eetgedrag van de jongeren onderzocht en hoe ze tegen voeding aankijken. Het tweede onderdeel ging in op de groenteconsumptie en de groentebeleving van de jongeren en de factoren die daarop van invloed zijn. Het laatste gedeelte van de vragenlijst was een keuze-experiment waarbij de jongeren gevraagd werd te kiezen tussen twee of drie groenteproducten en deze te beoordelen op een aantal productkarakteristieken.

The increases in obesity prevalence coincide with changes in our food environment, such as an increased consumption of processed, energy dense foods. This suggests that the foods we consume are at least partly responsible for the obesity epidemic. The aim of this thesis is therefore to investigate food characteristics, with the focus on taste, eating rate and energy density, and their relation to dietary intake. Taste is studied in two respects. First, the contribution of taste qualities to the diet is investigated, using the Food Consumption Survey 2003. Foods are classified according to their predominant taste (sweet, salty or savoury, sour, bitter or neutral). Energy intake of the foods within taste categories is assessed, showing that the largest part (34%) of the daily energy intake originates from sweet foods. Second, it is investigated whether taste, which is supposed to be a nutrient sensor, can fulfil this function within the current diet. Intensities of the five basic tastes of 50 commonly consumed foods are therefore assessed and associated with the nutrient content. Positive associations are found between sweetness and mono- & disaccharides and between both saltiness and savouriness and sodium and protein. The associations are less pronounced in highly processed foods, which suggests that in these foods the ability to sense nutrient content based on taste is limited. The influences of an incongruence between sensory properties and nutrient content are also investigated, byexamining the effects of fat perceptionon energy intake. We demonstrated that energy intake is almost 10% lower in case of visible fats compared to hidden fats, suggesting that hidden fats may contribute to overconsumption. Eating rateseems to be associated with food intake. The contribution of eating rate to the diet is investigated, using the Food Consumption Survey 2003. Foods are classified into one of four eating rate categories, and energy intake of the foods within each category is assessed. Results demonstrate that foods with slow calories (kJ/min) provide 10%, whereas foods with fast calories provide 37% to the daily energy intake. So in the current diet, the consumption of foods with a high eating rate is high. The effects of eating rate on intake are also investigated, showing that eatingrate is positively associated with food and energy intake. People may therefore be at risk of overconsumption, when consuming foods with a high eating rate. Consuming energy dense snacks is often blamed for affecting energy balance, but findings are inconclusive. Therefore, effects of snack consumption on body weight are investigated. No changes in body weight are observed after 8 weeks, when energy density of snacks was either low or high. This suggests that consuming snacks does not necessarily contribute to weight gain, at least in normal-weight young adults. In conclusion,when taste and other sensory properties do not accurately reflect the nutrient content, which applies particularly to highly processed foods, this may lead to high food intakes. In addition, a large part of the daily energy intake originates from foods with a high eating rate, which stimulates food and energy intake. So the high eating rate of the foods in the current diet may be responsible for overconsumption. These findings may be helpful in following the recommendations of the Nutrition Centre to lose weight. Last, even though we did not find evidence that consuming energy dense snacks results in weight gain, the advice should nevertheless be to limit the intake of energy dense foods, at least until evidence becomes more conclusive.

Background and aim The sensory attributes of food play a key role in the selection and termination of meals and their rewarding properties. The majority of our foods are either sweet or savoury tasting. In addition, within our food range, savoury-tasting foods contain in general higher levels of protein. The effect of specific taste modalities on human food intake, however, requires further clarification. The primary aim of this thesis was to investigate the role of sweet and savoury taste in food intake and food preferences. The secondary aim was to provide more insight into the processes of explicit and implicit liking and wanting, to be able to identify underlying reward mechanisms involved in food intake behaviour. Methods We conducted series of experiments where healthy young adults participated. We started by investigating the difference between a sweet and savoury taste on satiation, independent of palatability, texture, energy density, and macronutrient composition (n=64). Next, the effect of sweet and savoury taste of a single meal on subsequent satiety and food preferences was investigated (n=61). To further explore the effect of taste in the context of a complete diet on satiety and food preferences, the effect of three 24-h diets that differed only in taste (predominantly sweet tasting, predominantly savoury tasting, or a mixture of sweet and savoury tasting) were compared (n=39). Next, we separated the influence of taste from within-meal protein content on satiety and food preferences, by comparing the effect of sweet and savoury high and low protein single meals (n=60). Finally, the effect of long-term protein status on satiety and food preferences was investigated by comparing the effect of two 14-d diets that differed in protein content (a low protein diet vs. a high protein diet) (n=37). Results Sweet and savoury taste, independent of palatability, texture, energy density, and macronutrient composition, did not differ in their effect on satiation and satiety in terms of subsequent ad libitum intake. Sweet and savoury taste did differ in their effect on subsequent food preferences. In general, after eating a food with a certain taste, appetite for foods with a similar taste was lower than for foods with a dissimilar taste, hence, a clear transfer effect of sensory specific satiety was demonstrated. This transfer effect was not equipotent for sweet and savoury taste; after eating a sweet single meal or sweet 24-h diet, preferences for sweet and savoury foods did not differ. Eating a savoury single meal or savoury 24-h diet, however, led to a clear preference for sweet foods. Neither sweet or savoury tasting single meals nor sweet or savoury 24-h diets shifted food preferences towards high or low protein foods. It was shown that protein content of a meal, independent of taste, did not have an effect on satiety and food preference. We did observe, however, an effect of protein status: after a 14-d low protein diet, there was an increase in ad libitum protein intake, compared to after a 14-d high protein diet, while total energy intake was not different. In addition, food preference for savoury high protein foods was increased. Regarding the different components of food reward it was demonstrated that in all studies both explicit and implicit measures correlated with several aspects of eating. It appeared that in a controlled setting, i.e. in the sensory booths, explicit processes played a stronger determining role in satiation (meal size) than implicit processes. Food choices appeared to be made on a more unconscious level. In a setting where subjects could behave more naturally (i.e. self-selection and serving of foods in a relaxed environment where subjects could sit and eat together), implicit, unconscious processes seemed to explain food intake behaviour more than explicit processes. When subjects experienced protein shortage, after the 14-d low protein diet, it appeared that implicit processes of wanting played a stronger determining role in decisions about what to eat. Conclusion Sweet and savoury taste do not differ in their effect on satiation or satiety in terms of subsequent ad libitum intake. The taste of a meal or diet does have a large effect on subsequent food preferences, thereby showing a clear transfer effect which is not equipotent for sweet and savoury taste. Savoury taste exerts a stronger modulating effect on subsequent food preferences than sweet taste. Sweet and savoury taste of a single meal or 24-h diet do not differ in their effect on food preferences for high or low protein foods. In addition, within-meal protein content seems not to influence satiety or food preferences. A low protein status, however, through selective reduction of dietary protein intake, elicits compensatory changes in food intake and food preferences to restore adequate protein status. It appears that both conscious (explicit) and unconscious (implicit) processes are involved in satiation and food choice. The role implicit motivational processes play in driving food choice is not static, but appears to vary. This is especially the case when homeostasis is challenged (by depleting macronutrient stores), where implicit processes of wanting appear to play a stronger determining role in decisions about what to eat.

Background and aim The rise in obesity over the last decades is considered to be related to changes in the food environment. Our current diet exists of foods that facilitate fast intake of energy and minimal oral processing. Various studies showed that higher eating rate leads to higher food intake, and therefore promote energy overconsumption. When consuming at a high eating rate, the food spends less time in the oral cavity, resulting in less sensory exposure per gram food. The exposure to the taste of the food in the oral cavity is potentially important in controlling food intake. The studies in this thesis investigated the principle mechanisms through which orosensory exposure affects satiation. The factors that were studied were taste intensity, oral residence duration and bite size. The impact of these factors and their relative contributions to satiation will provide tools for designing new foods to prevent overconsumption. Methods We conducted five studies. The subjects that participated in the studies were healthy young normal weight adults. Satiation was measured by ad libitum intake and subjective ratings of hunger and fullness. Tomato soup was used as test product in all studies. We started by investigating the effect of taste intensity on ad libitum intake (n=48). Salt was used to vary the taste intensity in soup. We selected two salt concentrations for low-salt and high-salt soup that were similar in pleasantness on an individual basis. In the next study, salt taste intensity in soup was investigated again, but this time we changed the state of hunger (a preload was offered) and the meal composition (subjects were served a second course after the soup) (n=43). In the third study, the impact of taste intensity versus the duration of orosensory exposure (manipulated by changing the bite size) on satiation was investigated, by using peristaltic pumps to control the bites (n=55). The fourth study focussed on the underlying mechanisms of bite size on food intake (n=56). Therefore, separate effects of oral residence duration per gram food and number of bites per gram food on ad libitum intake were assessed. Finally, we investigated if bite size affects the perceived food intake. Subjects estimated the amount consumed after intake with small or large bites, in both focussed and a distracted states (n=53). In addition, effects of distraction on bite size were investigated. Results Taste intensity did not affect ad libitum intake when the soup was presented as single lunch-item in a hungry state. However, higher taste intensity reduced ad libitum intake by ~8%, when the soup was presented after a preload or as a starter followed by a second meal. Smaller bite sizes decreased ad libitum intake by ~25% and did not interact with taste intensity. That smaller bites are more satiating than larger bites was confirmed by hunger and fullness ratings. Hunger decreased faster per consumed gram food when consuming with small bites compared to large bites. A similar effect was found for the increase in fullness. Ad libitum intake was separately reduced by longer oral residence duration and higher number of bites per gram food, there was no interaction between the two variables. Time-intensity measurements showed that both higher number of bites and longer oral residence duration increase the total magnitude of orosensory exposure to the taste of the food. Consumption with large bites resulted in underestimations of the amount consumed, whereas consumption with small bites did not. Distraction increased ad libitum intake. Distraction led to a higher number of bites over the meal but did not affect bite size. Conclusions This thesis demonstrates that consuming foods with smaller bite sizes, longer oral residence durations and higher taste intensities lowers food intake. These effects are possibly explained through their enhancement of the orosensory exposure to the taste of the foods. Consumption with large bites leads to underestimation of the amount that is consumed. An underestimation of the amount consumed is a risk factor for overconsumption. These results could be used by the food industry to enhance the satiating capacity of foods in order to prevent overconsumption and decrease the prevalence of obesity.

In 2009 a survey was conducted among a representative sample of around 500 young people between the ages of 12 and 18 in the Netherlands. The first section examined the eating patterns of the young people and their attitude towards nutrition. The second section examined their vegetable consumption and how they experience vegetables, as well as the factors which influence this. The final section consisted of a choosing experiment, in which the participants were asked to choose from among two or three vegetable products and evaluate them on the basis of a number of product characteristics.

The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

In this report a Dynamic Energy Budget (DEB) model for razor clams (Ensis directus) is presented. A DEB model is a generic model describing growth and development of individual organisms as a function of environmental conditions. The DEB model for Ensis directus is based on the standard DEB model. The species specific primary DEB parameters are estimated with the Add_my_pet procedure, using literature data and the results of laboratory experiments with Ensis directus. The performance of the model is demonstrated by theoretical model experiments with varying environmental conditions. In following projects, the model will be used to predict and quantify the effects of sand mining on the shellfish community in the Dutch coastal zone. For this purpose the functional response of the model should be adapted so that the combined effect of changing phytoplankton and suspended sediment concentration on the uptake rate can be simulated.

In this report a Dynamic Energy Budget (DEB) model for razor clams (Ensis directus) is presented. A DEB model is a generic model describing growth and development of individual organisms as a function of environmental conditions. The DEB model for Ensis directus is based on the standard DEB model. The species specific primary DEB parameters are estimated with the Add_my_pet procedure, using literature data and the results of laboratory experiments with Ensis directus. The performance of the model is demonstrated by theoretical model experiments with varying environmental conditions. In following projects, the model will be used to predict and quantify the effects of sand mining on the shellfish community in the Dutch coastal zone. For this purpose the functional response of the model should be adapted so that the combined effect of changing phytoplankton and suspended sediment concentration on the uptake rate can be simulated.

Background Despite a large amount of research in the past decades, the role of polyunsaturated fatty acids (PUFA) in the prevention of coronary heart disease (CHD) and stroke is still debated. Inconsistent findings in epidemiological studies may be due to methodological limitations of dietary assessment, which could be overcome by using PUFA levels in blood as a biomarker of intake. This thesis investigates dietary intake and plasma levels of various n-6 and n-3 PUFA in relation to CHD and stroke within a population-based sample in the Netherlands. Methods The associations of dietary intake of PUFA (assessed by food-frequency questionnaire) with incident CHD and stroke were examined in cohort studies. PUFA levels in plasma cholesteryl esters were measured in nested case-control studies. N-6 PUFA included linoleic acid and arachidonic acid and the n-3 PUFA included the marine-derived eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the plant-derived alpha-linolenic acid (ALA). We used the “Monitoring Project on Cardiovascular Disease Risk Factors“ and the “Monitoring Project on Risk Factors for Chronic Diseases” (MORGEN study), two large similar population-based cohorts with baseline measurements in 1987-1997 and follow-up for CHD and stroke incidence. Additionally, we performed a meta-analysis of prospective epidemiological studies on PUFA in cholesteryl esters and CHD risk. Results A 4-5 en% difference in linoleic acid intake was not associated with incident CHD, whereas plasma linoleic acidwas inversely, but statistically non-significantly, associated with fatal CHD. In the meta-analysis, a 5% higher plasma linoleic acid level was related to a significant 9% lower CHD risk. Both ALA intake and status were not associated with CHD. The top quartiles of EPA-DHA (~250 mg/d) and fish intake (~1 fish meal/week) were related to a ~50% lower risk of fatal CHD compared to the bottom quartiles. However, this was not confirmed in plasma EPA-DHA. An ALA intake ≥1.1 g/d was associated with a 35-50% lower stroke incidence, compared with lower intakes. In women, but not in men, a significantly inverse relation was observed for EPA-DHA and fish intake with incident stroke, with a ~50% lower risk in the top quartile compared with the bottom quartile. Plasma PUFA levels were, however, not related to incident stroke. Conclusion The hypothesis of a beneficial effect of linoleic acid on CHD was confirmed in our biomarker study, but not in the study that used dietary intake data. For EPA-DHA, on the other hand, dietary intake was inversely related to fatal CHD and incident stroke, whereas cholesteryl ester EPA-DHA were not associated. The same applied to ALA intake in relation to incident stroke. Inconsistencies between PUFA intake and status with cardiovascular diseases could be attributed to the limited range of variation in PUFA intake in combination with measurement error.