Glossogyne tenuifolia (GT) is a native perennial plant growing across the coastline areas in Taiwan. The current study aimed to examine the efficacy of GT extract in ameliorating physical fatigue during exercise and increasing exercise performance. Fifty male Institute of Cancer Research (ICR) mice were randomly segregated into five groups (n = 10) to GT extract orally for 4 weeks, at different concentrations (50, 100, 250, and 500 mg/kg BW/day): LGT 1X, MGT 2X, HGT 5X, and HGT 10X groups. Forelimb grip strength, endurance swimming time, serum biochemical marker levels, blood lipid profile and histological analysis of various organs were performed to assess the anti-fatigue effect and exercise performance of GT extract. The forelimb-grips strength and endurance-swimming time of GT-administered mice were increased significantly in a dose-dependent manner when compared to the control. Serum glucose, creatine kinase, and lactate levels were increased significantly in the HGT 10X group. Liver marker serum glutamic-oxaloacetic transaminase (GOT) was increased in the HGT 5X and HGT 10X groups, whereas Serum Glutamic Pyruvic Transaminase (GPT) was not altered. Renal markers, creatinine and uric acid levels, were not altered. Muscle and hepatic glycogen levels, which are essential for energy sources during exercise, were also significantly increased in a dose-dependent manner in all GT extract groups. No visible histological aberrations were observed in the vital organs after GT extract administration. The supplementation with GT extract could have beneficial effects on exercise performance and anti-fatigue function without toxicity at a higher dose.