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To gain better insight into the potential health effects of fruits and vegetables, reliable biomarkers of intake are needed. The main purpose of this study was to investigate the ability of flavonoid excretion in both 24-h and morning urine samples to reflect a low intake and moderate changes in fruit and vegetable consumption. Furthermore, the urinary excretions of 4-pyridoxic acid (4-PA) and potassium were investigated as other potential biomarkers of fruit and vegetable intake. The study was designed as a 5-d randomized, controlled crossover study. On d 1-3, the men (n = 12) consumed a self-restricted flavonoid-free diet. On d 4, they were provided a strictly controlled diet containing no fruits or vegetables (basic diet). On d 5, they consumed the basic diet supplemented with 300 or 600 g of fruits and vegetables. The total excretion of flavonoids in 24-h urine samples increased linearly with increasing fruit and vegetable intakes (r(s) = 0.86, P < 1 x 10(-6)). The total excretion of flavonoids in morning urine also increased, but the association was weaker (r(s) = 0.59, P < 0.0001). Urinary 4-PA in 24-h and morning urine samples increased significantly only with the 600-g increase in fruit and vegetable intake, whereas the excretion of potassium in urine did not reflect the changes in fruit and vegetable intake. We conclude that the total excretion of flavonoids in 24-h urine may be used as a new biomarker for fruit and vegetable intake.