Abstract: In this study, a fatigue mouse model was developed to investigate the effects of different doses of pumpkin seed peptide on fatigue in mice. Forty-five mice were randomly assigned to five groups: a normal control group, a model group, and three pumpkin seed peptide groups with low, medium, and high-doses. First, the amino acid composition of the pumpkin seed peptide was analyzed. Second, the changes in swimming time, serum harmful substances, energy substances, oxidative stress, and inflammation levels in mice were examined following intervention with different doses of pumpkin seed peptide. The results revealed that the pumpkin seed peptide contained 18 amino acids, including Glu, Leu, and Pro. Animal experiments demonstrated that compared to the model group, swimming time, muscle glycogen, and hepatic glycogen levels were significantly increased (P<0.05), while serum urea nitrogen, lactic acid, and lactate dehydrogenase levels were significantly reduced (P<0.05) in mice treated with a high dose of pumpkin seed peptide. Additionally, the high-dose group exhibited significantly greater improvements (P<0.05) in the aforementioned indices compared to the low- and medium-dose groups, except for liver glycogen and lactate dehydrogenase levels. Furthermore, in the high-dose pumpkin seed peptide group, the antioxidant enzymes SOD, GSH-Px, and CAT were significantly elevated (P<0.05) in both serum and liver tissues, whereas MDA levels were significantly reduced (P<0.05). Analysis of inflammatory markers revealed that pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) were significantly reduced (P<0.05), while anti-inflammatory cytokines (IL-4 and IL-10) were significantly elevated (P<0.05) compared to the model group. Correlation analysis further revealed that Trp and Leu exhibited significant negative correlations (P<0.05) with pro-inflammatory cytokines and serum markers (lactate dehydrogenase and urea nitrogen). In conclusion, pumpkin seed peptide demonstrates anti-fatigue properties, with high-dose administration exerting the most pronounced effects. This study provides a theoretical foundation for the application of pumpkin seed peptide in fatigue management.