International audienceWhen the mother’s own human milk is unavailable or limited, pasteurized human milk from milk banks is preferentially administered instead of infant formula, especially for vulnerable hospitalized neonates. Holder pasteurization (62.5 °C, 30 min) may alter human milk composition and structure, which may modulate its digestive behavior. An in vitro dynamic system was set up to simulate the gastrointestinal digestion of term newborns in order to compare the kinetics of lipolysis, proteolysis and structural disintegration of raw versus pasteurized human milk. Human milk from 5 donors was pooled. Half of the pool was either administrated raw (RHM) or pasteurized (PHM). Digestions were conducted at least in duplicate for RHM and PHM. Heat-induced protein aggregation was observed in PHM. During gastric digestion, β-casein was proteolyzed significantly faster for PHM than for RHM (p < 0.05), whereas lactoferrin tended to be proteolyzed slower (p = 0.07) for PHM. Pasteurization selectively affected the intestinal release of some amino acids. At any time of the gastrointestinal digestion, the lipolysis of PHM was significantly lower than that of RHM, but no impact was observed on the profile of released fatty acids. RHM presented a structural destabilization after 60 min of gastric digestion, while there was no large variation for PHM. In the intestinal phase, the evolution of the particle sizes was rather similar. Overall, Holder pasteurization impacted the proteolysis, lipolysis and disintegration of human milk. However, this impact was limited and the physiologic and metabolic consequences remain to be investigated