The purpose of this study was to determine whether surface electromyography (EMG) assessment of myoelectric manifestations of muscle fatigue is capable of detecting differences between the vastus lateralis and medialis muscles which are consistent with the results of previous biopsy studies. Surface EMG signals were recorded from the vastus medialis longus (VML), vastus medialis obliquus (VMO) and vastus lateralis (VL) muscles during isometric knee extension contractions at 60% and 80% of the maximum voluntary contraction (MVC) for 10s and 60s, respectively. Initial values and rate of change of mean frequency (MNF), average rectified value (ARV) and conduction velocity (CV) of the EMG signal were calculated. Comparisons between the two force levels revealed that the initial values of MNF for the VL muscle were greater at 80% MVC compared to 60% MVC (P<0.01). Comparisons between the vasti muscles demonstrated lower initial values of CV for VMO compared to VL at 60% MVC (P<0.01) and lower than VML and VL at 80% MVC (P<0.01). In addition, initial values of MNF were higher for VL with respect to both VML and VMO at 80% MVC (P<0.01) and initial estimates of ARV were higher for VMO compared to VML at both force levels (P<0.01 at 60% MVC and P<0.05 at 80% MVC). For the sustained contraction at 80% MVC, VL demonstrated a greater decrease in CV over time compared to VMO (P<0.05). These findings suggest that surface EMG signals and their time course during sustained isometric contractions may be useful to non-invasively describe functional differences between the vasti muscles.