Objective Oyster was used as the raw material to prepare antioxidant oyster peptides by hydrolysis with complex proteases (papain, alkaline protease, and aminopeptidase), and their anti-exercise fatigue activity was evaluated.Methods Using degree of hydrolysis and DPPH radical scavenging rate as evaluation indicators, response surface methodology was applied to optimize the preparation process of oyster peptides, and the fatigue-alleviating effects of oyster peptides were investigated in exercise-induced mice.Results The optimal extraction conditions for oyster peptides were as follows: 4% complex protease addition, hydrolysis pH 7.5, material-to-liquid ratio 1∶7.5 (g/mL), hydrolysis temperature 57 ℃, and hydrolysis time 7 h. Under these conditions, the degree of hydrolysis was 48.3%, and the DPPH radical scavenging rate was 60.42%. The hydrophobic amino acid content in oyster peptides was 33.18 g/100 g, accounting for 48.28% of total amino acids. The molecular weight was mainly concentrated below 5 000, with low molecular weight components (<1 000) accounting for 71.45%, indicating that oyster peptides were mainly small peptides with good antioxidant capacity. In the anti-fatigue test in mice, compared with the blank control group, high-dose oyster peptide intervention significantly increased the fatigue time on the rotating rod and the weighted swimming time by 10.93 min and 13.34 min, respectively, indicating significantly improved exercise capacity (P<0.05). Meanwhile, oyster peptides significantly reduced blood lactate (BLA) and blood urea nitrogen (BUN) levels after exercise (P<0.05), and enhanced antioxidant levels in mice, with significant improvements in malondialdehyde (MDA) and superoxide dismutase (SOD) levels (P<0.05).Conclusion The oyster peptides prepared in this study exhibit good antioxidant activity, which helps enhance exercise endurance and alleviate exercise-induced fatigue in mice.