Background: Holder pasteurization has been reported to modify human milk composition and structure by inactivating bile salt–stimulated lipase (BSSL) and partially denaturing some of its proteins, potentially affecting its subsequent digestion. Objective: We sought to determine the impact of human milk pasteurization on gastric digestion (particularly for proteins and lipids) in preterm infants who were fed their mothers’ own milk either raw or pasteurized. Design: In a randomized controlled trial, 12 hospitalized tube-fed preterm infants were their own control group in comparing the gastric digestion of raw human milk (RHM) with pasteurized human milk (PHM). Over a 6-d sequence, gastric aspirates were collected 2 times/d before and after RHM or PHM ingestion. The impact of milk pasteurization digestive kinetics and disintegration was tested with the use of a general linear mixed model. Results: Despite inactivating BSSL, instantaneous lipolysis was not affected by pasteurization (mean 6 SD at 90 min: 12.6% 6 4.7%; P . 0.05). Lipolysis occurred in milk before digestion and was higher for PHM than for RHM (mean 6 SD: 3.2% 6 0.6% and 2.2% 6 0.8%, respectively; P , 0.001). Pasteurization enhanced the proteolysis of lactoferrin (P , 0.01) and reduced that of a-lactalbumin (only at 90 min) (P , 0.05). Strong emulsion destabilization was observed, with smaller aggregates and a higher specific surface for PHM (P , 0.05). Pasteurization did not affect gastric emptying (w30-min half time) or pH (mean 6 SD: 4.4 6 0.8) at 90 min. Conclusions: Overall, pasteurization had no impact on the gastric digestion of lipids and some proteins from human milk but did affect lactoferrin and a-lactalbumin proteolysis and emulsion disintegration. Freeze-thawing and pasteurization increased the milk lipolysis before digestion but did not affect gastric lipolysis. Possible consequences on intestinal digestion and associated nutritional outcomes were not considered in this study. This trial was registered at clinicaltrials.gov as NCT02112331.