ABSTRACT:Gypenosides (Gyp), the main component in Gynostemma pentaphyllum, exert various pharmacological activities. Although Gynostemma pentaphyllum has been reported to improve endurance exercise performance and delayed fatigue, the effect of Gyp on fatigue and the underlying mechanisms have not yet been illustrated. In the present study, we established a fatigue mice model induced by long‐term high‐intensity swimming to explore the potential effect of Gyp on peripheral and central fatigue. The results revealed that Gyp prolonged the exhaustive swimming time and improved fatigue associated parameters including blood GLU, LA, NH3, LDH, HG and MG, demonstrating anti‐fatigue effects. In EF mice, Gyp supplementation decreased IL‐6, TNF‐α, MDA while increased SOD both in the muscle, liver, colon and hippocampus, showing anti‐inflammatory and anti‐oxidative effects. Furthermore, Gyp upregulated ZO‐1, Occludin and Claudin‐1 expressions in the colon, thereby maintaining the integrity of the intestinal barrier to inhibit inflammation. Gyp decreased 5‐HT content while increased DA in the frontal cortex, exhibiting a regulation on the neurotransmitters. In conclusion, Gyp alleviated the peripheral and central fatigue via anti‐inflammation, anti‐oxidation on the muscle, liver, colon, and neurotransmitter regulation on the hippocampus and frontal cortex, respectively. The detailed mechanisms at the cellular and molecular level remains to be elucidated in the future.