The purpose of this study was to identify and compare the Electromyographic Fatigue Threshold (EMG(FT)) determined in the Vastus Lateralis (VL), Rectus Femoris (RF), Biceps Femoris (BF), Semitendinosus (ST) and Tibialis Anterior (TA) during stationary cycling in trained cyclists and non-cyclists. Using a cycle ergometer, 13 cyclists (28.4 +/- 6.9 years; 70.3 +/- 13 kg; 176.1 +/- 8.5 cm) and 11 non-cyclists (25.8 +/- 4 years; 73 +/- 9.1 kg; 175 +/- 6.4 cm), performed a maximum incremental test (ITmax) (90 rpm) to determine the (EMG(FT)). Maximal power output (W(PEAK)) reached by cyclists was higher than for non-cyclists (372.6 W and 248.9 W respectively) (P < 0.01). For the five muscles analyzed in cyclists, EMG(FT) occurred at 85.7% of cases in the VL, 92.9% in RE 78.6% in BE 78.6% in ST and 50% in TA, while in the non-cyclists group, this occurrence was 100% to muscle VL, 100% to RF, 92.6% to BF, 78.6% to ST, and 78.6% to TA. Analyzing the percentage corresponding to the power at EMG(FT) in relation to W(PEAK) reached, no differences between groups were observed for RF, BF and ST, however VL and TA, as well as the mean from all muscles were lower for cyclists than non-cyclists (P < 0.05). The present results showed that EMG(FT) is more easily identified in RF and VL muscles for both groups, and it may be an interesting method to evaluate the adaptive responses from aerobic and anaerobic metabolisms during cycling training programs.