Objectives and Study Supplementing human milk (HM) is often necessary to fulfill the nutritional needs of premature infants. This study aimed to determine if enriching HM with either an experimental donkey milk-derived fortifier (DMF), containing whole DM proteins, or a commercial bovine milk-derived fortifier (BMF), containing hydrolyzed BM whey proteins, affects the release of peptides during digestion. Methods Milk samples were collected, fortified and digested by means of DIDGI® bi-compartmental in vitro dynamic system to simulate the digestion of a preterm newborn at a postnatal age of four weeks. Identification and quantification of the peptides in undigested and digested samples was performed by HPLC-HRMS. Each identified peptide was quantified by means of label-free MS, using MassChroQ software. Data were elaborated by Multivariate analyses of variance (Principal Component Analysis - PCA). Results Results indicated that the different fortifiers did not significantly alter the overall intensity of HM peptides. However, fortification led to distinct impacts on the release of specific bioactive peptides. Additionally, when HM was supplemented with DMF, there was a minor delay in the release of peptides from lactoferrin and ?-lactalbumin. Conclusions Lactoferrin from human milk (HM) exhibited less peptide release during intestinal digestion when fortified with the DM-derived fortifier. This prolonged presence of intact lactoferrin holds promise for bolstering gut protection against infections and inflammation. Another benefit of fortifying HM with the DM-derived fortifier is the generation of extra anti-inflammatory peptides from DM during digestion. These discoveries carry significant implications for the clinical nutrition of premature infants, who frequently face challenges related to intestinal health issues and inflammation