Physical fatigue is one of the important problems that plague public health. Our objective is to investigate the anti-fatigue effect of a botanical beverage with ginseng extract and pueraria extract, including ginseng, wolfberry, Polygonatum, Pueraria, and niacin. Sixty ICR male mice were randomly divided into 6 groups, namely blank control group, experimental control group (swimming group), positive control group (caffeine group), low-dose group (3.33 g/kg BW), medium-dose group (6.67 g/kg BW), and high-dose group (13.33 g/kg BW) of botanical beverage. Then we performed behavioral examination, including grip and turn-club test, and investigated fatigue-related biochemical indexes and behavioral tests after consecutive gavage administration for 30 days.. Results showed that the fatigue rotarod time and forelimb pulling force of the mice are significantly increased in the middle and high dose groups of botanical beverage and the caffeine group, compared with the blank control group. The contents of blood sugar level, liver glycogen, and muscle glycogen of the mice in the middle dose group increased by 53.58%, 18.75%, and 45.00%, respectively, compared with the swimming group. While the contents of blood lactate and urea nitrogen and the activities of lactate dehydrogenase and creatine kinase in the middle-dose group decreased by 5.56%, 18.78% and 27.18%, 9.45%, respectively. It’s concluded that botanical beverage with ginseng extract and pueraria extract had the effect on relieving physical fatigue.