Abstract: To optimize the purification process condition of total polyphenols of extract from Cistanche by macroporous resin, the HPD-400 macroporous resin type was chosen through static adsorption-desorption experiments, meanwhile, isothermal adsorption model and adsorption kinetics model of the resin were furthermore investigated and then anti-motor fatigue of animal experiment was carried out. The results showed that equilibrium adsorption data of HPD-400 macroporous resin for total polyphenols were fitted Langmuir isotherm equation, the adsorption decrease with the increasing of temperature, and the kinetics fitted the quasi-second-order kinetics equation. The optimum purification process conditions were as follows:the initial concentration in sample solution 6 mg/mL, flow rate 2 mL/min, loading sample volume 60 mL, and 60% (v/v) ethanol 180 mL was used as eluent with flow rate of 1 mL/min. After purification, the purity of total polyphenols in product was increased from 20.12% to 42.63%. Compared with the blank control group, the groups of polyphenols of Cistanche at all doses significantly prolonged weight-loaded swimming time of mices (P<0.05, P<0.01), obviously improved activity of lactic dehydrogenase and storage of hepatic glycogen and muscle glycogen in vivo (P<0.05, P<0.01), decreased lactic acid level after sporting (P<0.01).Therefore, polyphenols of Cistanche could enhanced sporting stamina and had good resisting movement fatigue effect, the study could provide reference for its subsequent development and exploitation.