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Cheese is an important source of protein in the human diet, and its digestibility depends on its macro and microstructure. This study investigated the effect of milk heat pre-treatment and pasteurization level on the protein digestibility of produced cheese. An in vitro digestion method was used considering cheeses after 4 and 21 days of storage. The peptide profile and amino acids (AAs) released in digestion were analyzed to evaluate the level of protein degradation following in vitro digestion. The results showed the presence of shorter peptides in the digested cheese from pre-treated milk and 4-day ripening while this trend was not observed after 21 days of storage, showing the effect of storage period. A significantly higher content of AAs was found in digested cheese produced from milk subjected to a higher temperature of pasteurization, and there was a significant increase in total AA content in the cheese after 21 days of storage, confirming the positive effect of ripening on protein digestibility. From these results emerges the importance of the management of heat treatments on the digestion of proteins in soft cheese.