The corn gluten meal, byproduct through the corn starch industry, was hydrolyzed with the combination of alkaline protease and papain to produce oligopeptides with high Fisher-values. Response surface methodology was applied to optimize the hydrolysis conditions, including reaction temperature, pH, enzyme to substrate ratio, and percentage papain added. The highest degree of hydrolysis (29.51%) was obtained at a reaction temperature of 55?C, a reaction pH at 11.10, an enzyme to substrate ratio of 0.85, and percentage of papain added at 20%, which was in agreement with the predicted value (30.84%) estimated by response surface methodology within a 95% confidence interval. In addition, the final product was the mixture of oligopeptides with relatively high content of branched-chain amino acids and in vivo the effect on physical fatigue was investigated by using a loaded swimming test, along with the determination of blood lactic acid, blood urea nitrogen, liver glycogen and muscle glycogen contents. The data showed that the oligopeptides could extend exhaustive swimming times of mice, as well as increase liver and muscle glycogen contents and decrease blood lactic acid contents. These results support that the oligopeptides, prepared from corn proteins using the combination of two proteases, have an anti-physical fatigue effect.