This study examined the EMG activities of the medial and lateral vasti muscles with four different surface electrode positions during isometric knee extension and knee perturbation.Repeated measures design.Orthopaedic rehabilitation laboratory of a university.Eight able-bodied non-athletic male volunteers.The relative onset time difference in milliseconds of vastus medialis obliquus (VMO) and vastus lateralis (VL) during knee perturbation, and the normalized EMG amplitude in root-mean-square ratio of VMO:VL during submaximal isometric knee extension were analyzed using repeated measures ANOVA.Different electrode positions on VL resulted in different VMO-VL onset time (p=0.002) and normalized VMO:VL activity ratios (p=0.002, 0.014).The position of surface electrodes has significantly affected the EMG readings of the vasti muscles. This finding has vital clinical implications for the application of EMG measurement and biofeedback training for the rehabilitation of the vasti muscles.