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Purpose: This study aims to achieve a better understanding of the trend of maternal milk macronutrient levels by analyzing protein, lipid, carbohydrate, and energy content of the breast milk and investigate the possible confounders of macronutrient content during the first 4 weeks of lactation to meet the specific needs of babies, guide optimum fortification of maternal milk. Patients and Methods: Breast milk from 39 mothers who had delivered preterm infants and 21 mothers of term infants were collected longitudinally for the first 4 weeks of lactation. Fresh milk samples were obtained on day 3, 7, 14, and 28 of lactation. The samples are analyzed using mid-infrared milk analyzer (MIRIS Human Milk Analyzer, HMA; Miris AB, Uppsala, Sweden). Results: Colostral milk protein concentrations of mothers of both preterm and term infants were significantly higher and the protein content of the samples decreased in time, according to the week of lactation during the study period. In contrast, fat, carbohydrate, and energy content were lowest in the colostral milk in both groups. When preterm and term milks were compared, fat and carbohydrate levels on 28th day and energy levels on 14th and 28th days were significantly higher in term milks, whereas no difference in protein contents were observed between the two groups on each time period. Mode of delivery was found to have statistically significant correlation with protein content of the milk. Conclusion: This longitudinal study revealed significant changes in analyzed macronutrient contents of mother's milk over the first 4 week period.