This study systematically evaluated the anti-fatigue efficacy of high Fischer ratio oligopeptides (HFOs) from Antarctic krill (HFOs-AK) and explored its underlying mechanisms in a mouse model of exercise-induced fatigue. The results showed that HFOs-AK dose-dependently prolonged endurance swimming time, reduced metabolite levels (lactic acid, blood urea nitrogen, and blood ammonia), increased blood glucose, muscle glycogen, and liver glycogen, and decreased lactate dehydrogenase and creatine kinase levels. HFOs-AK also enhanced Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities and increased ATP content in muscle tissue. Additionally, HFOs-AK demonstrated significant antioxidant properties by increasing superoxide dismutase and glutathione peroxidase activities and reducing malondialdehyde levels. Further analysis revealed that HFOs-AK regulated energy metabolism and exerted anti-fatigue effects by activating the AMPK signaling pathway and up-regulating the expression of p-AMPK and PGC-α proteins. These findings suggest that HFOs-AK can be used as an auxiliary functional dietary molecule to enhance anti-fatigue activity and be applied in anti-fatigue functional foods.This study systematically evaluated the anti-fatigue efficacy of high Fischer ratio oligopeptides (HFOs) from Antarctic krill (HFOs-AK) and explored its underlying mechanisms in a mouse model of exercise-induced fatigue. The results showed that HFOs-AK dose-dependently prolonged endurance swimming time, reduced metabolite levels (lactic acid, blood urea nitrogen, and blood ammonia), increased blood glucose, muscle glycogen, and liver glycogen, and decreased lactate dehydrogenase and creatine kinase levels. HFOs-AK also enhanced Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities and increased ATP content in muscle tissue. Additionally, HFOs-AK demonstrated significant antioxidant properties by increasing superoxide dismutase and glutathione peroxidase activities and reducing malondialdehyde levels. Further analysis revealed that HFOs-AK regulated energy metabolism and exerted anti-fatigue effects by activating the AMPK signaling pathway and up-regulating the expression of p-AMPK and PGC-α proteins. These findings suggest that HFOs-AK can be used as an auxiliary functional dietary molecule to enhance anti-fatigue activity and be applied in anti-fatigue functional foods.