Fatigue is commonly marked by reduced endurance and impaired function, often linked to overexertion and chronic conditions. Quinoa (Chenopodium quinoa Willd.), with its rich amino acids and resilience to harsh conditions, offers a novel strategy for combating fatigue. This study explored the anti-fatigue effects of quinoa protein (QPro) and its hydrolysate (QPH) in weight-loaded swimming mice. After four weeks of oral administration, QPro and QPH significantly prolonged swimming duration, reduced serum fatigue biomarkers (lactic acid, urea nitrogen, lactate dehydrogenase, creatine kinase), and elevated glycogen reserves in the liver and muscle. RT-qPCR analysis indicated that QPH activated hepatic gluconeogenesis via G6Pase and PEPCK signaling and enhanced mitochondrial function through PGC-1α/NRF1/TFAM signaling in muscle. Additionally, QPro and QPH boosted antioxidant defenses by improving antioxidant enzyme activity, reducing malondialdehyde through the Nrf2/HO-1 pathway, and suppressing inflammation by reducing TNF-α and IL-6 levels. Network pharmacology identified 31 key targets involved in energy metabolism and inflammation, providing novel insights into the molecular mechanisms underlying the anti-fatigue properties of quinoa peptides. These findings highlight the potential of QPro and QPH as natural and bioactive ingredients in functional foods for enhancing endurance and mitigating fatigue.