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A two-stage in vitro digestion model system (a pepsin treatment for 2h followed by a pancreatin treatment for 2h, both at 37°C) was used to simulate the process of human gastrointestinal (GI) digestion to determine the changes in antioxidant activities of loach peptide previously prepared by papain digestion. Results showed that the final GI digests contained 38.1% free amino acids, with short chain peptides (<500Da) making up the rest of the biomass. Enzymatic breakdown of the GI digests increased their hydroxyl (12% increase), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (5% increase) radical scavenging activity, the reducing power (77% increase) and the chelating ability of Cu²⁺ (12% increase), compared to the blank. The results showed that the digestion by gastrointestinal proteases can be used to produce antioxidant peptides, with the advantage that the peptides …