Abstract: Fatigue represents a complex physiological and biochemical state within the body, and is a common sub-health condition in modern society. Prolonged fatigue can adversely affect health. In order to investigate the anti-fatigue efficacy of tuna peptides and their mechanism, this paper investigated the anti-fatigue effects of different molecular weight hydrolyzed peptides derived from tuna meat. The results indicated that when the total addition of animal enzyme and flavor enzyme reached 0.4%, with a ratio of 2:1, the hydrolysis degree of tuna meat peaked at 17.37%, and it received the highest sensory evaluation score of 9.78. Murine weighted swimming test indicated that, compared to the negative control group (NC), all groups of tuna peptides with varying molecular weights (Group Ⅰ: M_W>5 kDa, ATO-Ⅰ; Group Ⅱ: 5 kDa>M_W>3 kDa, ATO-Ⅱ; Group Ⅲ: M_W<3 kDa, ATO-Ⅲ) significantly extended the swimming duration and improved the biochemical markers in mice. The accumulation of lactate and urea nitrogen decreased, while the glycogen content in liver and muscle significantly increased (P<0.05). The activities of total superoxide dismutase in the serum increased by 9.51%, 7.74%, and 7.89% respectively; malondialdehyde levels were 6.92±0.14, 8.07±0.93, and 7.91±0.89 nmol/mL. The group ATO-Ⅲ demonstrated the most pronounced anti-fatigue effects. PCR experiments demonstrated that ATO could exert anti-fatigue effects by up-regulating genes such as adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferators-activated receptor γ coactivator 1alpha (PGC-1α). This research not only provides a scientific basis for the development of novel functional foods, but also lays a theoretical foundation for improving human health and developing anti-fatigue treatments.