In the frame of the COST action INFOGEST, a static in vitro digestion protocol has been elaborated aiming at the improvement of data comparability by harmonizing the experimental conditions. The success in harmonization was confirmed with inter-laboratory trials using skim milk powder as a standardized model food. Moreover, the physiological relevance of the gastric and intestinal endpoints of the static digestion protocol was demonstrated in a pig in vivo trial, with the same skim milk powder and samples collected from different sections of the digestive tract, as well as in a human study with from jejunal effluents. In vivo, digestion is a dynamic process influenced by peristalsis and by the gradual secretion of enzymes and juices and the dwell time of the food. To mimic these physiological mechanisms, dynamic in vitro digestion protocols are widely used. Until now, the differences of protein hydrolysis taking place during dynamic and static in vitro digestion have not been investigated. In this study, the gradual hydrolysis of the main milk proteins present in skim milk powder was digested with the dynamic DIDGI®-system using adult digestion protocol and the static harmonized INFOGEST method. Protein hydrolysis was analyzed by gel electrophoresis, peptide patterns were measured with mass spectrometry, and free amino acids with high pressure liquid chromatography. The peptide patterns at the gastric and intestinal endpoints of in vitro digestion showed a good approximation to the in vivo results from pigs. Moreover, gradual peptide generation was comparable in both in vitro digestion conditions. However, the dynamic protocol reflected the physiological situation better at the level of free amino acid release. Nonetheless, in both in vitro digestion protocols, absorption of free amino acids is not simulated, and they are therefore limited in reflecting the in vivo situation at this level.