Changes in substrate concentration were shown to affect the experimental maximum degree of hydrolysis (DHmax,exp). Such changes may affect the outcome of in vitro protein digestion models. To study this effect, apo α-lactalbumin (0.5–10%) was hydrolysed by digestive enzymes, bovine, porcine and human trypsins, bovine α-chymotrypsin or Bacillus licheniformis protease (BLP), at a constant enzyme to substrate ratio (E:S). Hydrolysis by human trypsin was not sensitive to changes in substrate concentration. The DHmax,exp by bovine and porcine trypsins, or bovine α-chymotrypsin increased with an increasing substrate concentration, while the DHmax,exp by BLP decreased. For hydrolyses with different DHmax,exp, a change in the percentage of intact protein versus DH was observed, meaning that the peptide concentrations differed during the hydrolyses. If some peptides could be protease inhibitors, the differences in DHmax,exp could be explained. This was confirmed by performing the hydrolysis at a higher E:S, which reached a higher DHmax,exp.