INTRODUCTION: We investigated 2 different methods for determining muscle fatigue threshold by electromyography (EMG). METHODS: Thirteen subjects completed an incremental treadmill running protocol for EMG fatigue threshold (EMGFT ) determination based on the critical power concept (EMGFT 1) and the breakpoint in the linear relationship between EMG amplitude and exercise intensity (EMGFT 2). Then, both the EMGFT 1 and EMGFT 2 were tested in a continuous treadmill running protocol. EMG was recorded from the rectus femoris (RF), vastus lateralis (VL), biceps femoris (BF), and lateral gastrocnemius (LG) muscles. RESULTS: For BF, EMGFT 2 was higher than EMGFT 1, and EMGFT 1 for BF was lower than EMGFT 1 for LG. EMG of RF was higher at EMGFT 2 than at EMGFT 1, and LG EMG was lower at EMGFT 2. CONCLUSIONS: EMGFT can be determined during a single treadmill running test, and EMGFT 1 may be the most appropriate method to estimate the muscle fatigue threshold during running.