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Human milk (HM) provides essential nutrition for ensuring optimal infant growth and development postpartum. Metabolomics offers insight into the dynamic composition of HM. Studies have reported the impact of lactation stage, maternal genotype, and gestational age on HM metabolome. However, the majority of the studies have considered changes within the first month of lactation or sampled with large intervals. This leaves a gap in the knowledge of progressing variation in HM composition beyond the first month of lactation. The objective of this study was to investigate whether the HM metabolome from mothers with term deliveries varies beyond 1 month of lactation, during the period in which HM is considered fully mature. Human milk samples (n = 101) from 59 mothers were collected at weeks 1–2, 3–5, 7–9, and 20–25 postpartum and analyzed using 1H nuclear magnetic resonance spectroscopy. Several metabolites varied over lactation and exhibited dynamic changes between multiple time points. Higher levels of HM oligosaccharides, cis-aconitate, O-phosphocholine, O-acetylcarnitine, gluconate, and citric acid were observed in early lactation, whereas later in lactation, levels of lactose, 3-fucosyllactose, glutamine, glutamate, and short- and medium-chain fatty acids were increased. Notably, we demonstrate that the HM metabolome is dynamic during the period of maturity.

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