Fatigue is a common phenomenon usually observed in healthy, as well as in nonhealthy, individuals that affects their performance and quality of life. Efficient supplementation to relieve fatigue is of significant importance. This study was designed to investigate the efficacy of three prescreened natural resources (Cervus elaphus L. [CEL], Angelica gigas Nakai [AGN], and Astragalus membranaceus Bunge [AMB]) against fatigue symptoms induced by heavy exercise. Effects on muscle fatigue and endurance capacity during exercise were investigated in C2C12 myoblasts and exercised mice. A combination of CEL, AGN, and AMB (CEL:AGN:AMB, 1:2:1) treatment in myoblasts reduced intracellular reactive oxygen species levels induced by hydrogen peroxide by ∼20 times (P < .001). The optimal mixture extract combination was determined as CEL:AGN:AMB, 1:2:1 (CAA), which was recombined by applying the extraction yield of individual substance for in vivo study. Compared to the exercise control (EC) group, the serum lactate dehydrogenase level decreased by ∼40% due to CAA administration. The proliferator-activated receptor gamma coactivator 1-alpha protein expression increased significantly (P < .05) after CAA administration compared to that observed in the normal control group. In parallel, CAA treatment significantly (P < .05) enhanced the maximum running time compared to the EC group. Overall, combinatorial administration exhibited greater efficacy compared to each individual treatment, indicating that CAA could be used as an efficient ergogenic and antifatigue supplement.