Abstract: The optimum purified technological process was confirmed on the basis of result of dynamic adsorption-desorption single-factor experiments. Blank control group(0.1 mL/g saline solution),positive control group(0.1 mg/g American ginseng)and the low,the medium and the high dosage groups of purified products(0.05,0.10,0.15 mg/g). After 30 d of continuous gavage,weight-bearing swimming tests were performed on each group of mice. The anti-fatigue effect of flavonoids of Radix Rehmanniae Preparata was investigated by measuring the weight-bearing swimming time,muscle glycogen(MG),hepatic glycogen(HG),serum urea nitrogen(BUN),lactic acid(LD).The results showed that the best purification process conditions were as follows:the initial concentration in sample solution was 8 mg/mL,loading flow rate was 2 mL/min,loading sample volume was 60 mL,the pH value of sample solution was 5,and the ethanol concentration was 80%(v/v),the eluent volume was 150 mL with flow rate of 1 mL/min. After purification,the purity of total flavonoids in product was increased from 2.25% to 12.18%. Compared with the blank control group,the groups of flavonoid of Radix Rehmanniae Preparata at all doses significantly prolonged weight-loaded swimming time of mice(P<0.05,P<0.01),obviously improved storage capacity of hepatic glycogen and muscle glycogen in vivo(P<0.05,P<0.01),and accelerated lactic acid metabolism(P<0.05,P<0.01),decreased urea nitrogen creation in serum after sport(P<0.05,P<0.01). Therefore,flavonoid of Radix Rehmanniae Preparata could enhanced sporting stamina in body.