Most studies assessing the macronutrient content of human milk are published retrospectively using analyzers that fail to determine sodium content and do not take into account the role of volume in milk composition. We aimed to describe macronutrient content and sodium content in human milk over time, observe any associations between them, and determine the factors associated with the evolution of milk composition. A prospective, longitudinal, monocentric study was undertaken. Contents of protein, fat, and lactose of 102 milk samples from 40 mothers were determined using a human milk analyzer and that of sodium with a flame spectrophotometer. Milk volumes along with clinical data were recorded. Protein content in the fourth quartile of volume was significantly lower than that in the first three, suggesting the existence of a volume threshold for protein content at approximately 445 mL. After multivariate analysis, it was found that maternal age, average volume, and lactation period remained significantly associated with protein content, maternal age remained significantly associated with fat content, and only average volume with sodium content. In consideration of previous findings along with our data, we suggest that extra care should be taken with fortification for feeding preterm infants when the mother’s milk volume is greater than 400–450 mL.

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