Although a number of studies have reported the anti-fatigue effect of anserine, its evidence seems to be still insufficient except the subjective evaluations by questionnaires. The aim of the present study was to propose a method to evaluate the anti-fatigue effect of anserine by a simple protocol and to verify the effectiveness of the method. Subjects were 17 healthy male volunteers (35.5 ± 5 yr., 75.5 ± 5.0 kg). They performed the isometric exercise tolerance test (ETT) on the rectus femoris muscle twice. Median frequencies (MDFs) of electromyogram and their regression curves were calculated for two ETTs. The angles between these curves were defined as a muscle fatigue index and were compared between anserine and water (control) intakes. The results indicate that anserine can alleviate muscle fatigue in humans. To the best of our knowledge, this is the first direct demonstration of the anti-fatigue effect of anserine on muscle in humans.