Purpose The purpose of this study was to examine the effects of interelectrode distance (IED) on the normalized electromyographic (EMG) amplitude and mean power frequency (MPF) from the vastus lateralis (VL) during a maximal, incremental cycle ergometer test. Methods Seventeen adults [mean +/- SD age = 24.1 +/- 2.9; VO2_max = 47.0 +/- 7.8 ml.kg−1·min−1] performed a maximal, incremental test to exhaustion on a cycle ergometer. Surface EMG signals from the VL were recorded during the final 10 seconds of each two minute power output using bipolar electrode arrangements with IEDs of 20, 40, and 60 mm. Polynomial regression analyses were used to determine the patterns of relationships for normalized (%max) EMG amplitude and MPF versus power output for each IED. In addition, one-way repeated measures ANOVAs were used to examine mean differences between the three IEDs for normalized EMG amplitude and MPF at a power output of 170 W. Results Polynomial regression analyses for each IED indicated that the normalized EMG amplitude (r = 0.86 - 0.87) and MPF (r = 0.30 - 0.36) versus power output relationships were best fit with quadratic models. Furthermore, there were no significant (P < 0.05) differences between the IEDs for normalized EMG amplitude or MPF at 170 W. Conclusions The results of this study indicated that interelectrode distances of 20, 40, and 60 mm resulted in the same pattern of responses for normalized EMG amplitude and MPF from the VL during a maximal, incremental test performed on a cycle ergometer.