Introduction: Donor human milk (DHM) represents the best alternative when mother’s own milk is not available, a common occurrence in Neonatal Intensive Care Units. Heat treatment of DHM is mandatory for safety reasons. Holder pasteurization (HoP, 62.5°C-30’) is recommended by all human milk bank guidelines. Recent studies have demonstrated that HoP affects the digestion profile and behavior of several human milk components. High Temperature-Short Time pasteurization (HTST, 71°C-15’’) is currently under evaluation as a promising alternative technology to limit the denaturation of some biological compounds of raw human milk. The aim of the present work was to assess whether the different types of pasteurization (HoP, HTST) impacted the digestive kinetics of human milk during in vitro dynamic digestion. Materials and Methods: pooled raw HM (RHM) was collected and processed by using the two pasteurization techniques. The pasteurized samples and RHM were digested in vitro using preterm gastrointestinal conditions. Samples were collected at different digestion times. Undigested and digested milks samples were characterized for their particle size distribution (PSD), triglyceride content, protein and amino acid (AA) profiles. Results: during gastric digestion, both pasteurization methods modified PSD, as compared to RHM. Caseins were rapidly hydrolyzed in the gastric phase unlike that for the whey proteins. Lactoferrin was hydrolyzed faster in the pasteurized samples in comparison to RHM, in which lactoferrin was resistant to gastro-intestinal digestion. Heat-treatments, consequently, affected the intestinal release of some AA, and a higher bioaccessibility of AA was found for HTST, as compared to HoP. Concerning lipolysis, at any time of the intestinal digestion phase, the lipolysis of HoP samples was significantly lower (p < 0.05) than in both RHM and HTST samples. Conclusions: this work provides the first important evidences on the differential impact of HoP and HTST pasteurization techniques on bioaccessibility of DHM nutrients and biological compounds, for preterm newborns.