Objectives: To investigate whether there was a gender difference in the intensity of electromyographic (EMG) activity in vastus medialis oblique (VMO) relative to vastus lateralis (VL). Design: A cross-sectional observational study measuring EMG activity during stepping down from a step and during straight leg raise exercises. Setting: University campus laboratory. Participants: Two groups of healthy participants were tested, one female (mean age 23.5 years, n = 15) and one male (mean age 23.5 years, n = 15). Main outcome measures: Surface EMG activity (sampling rate 1000 Hz) was recorded from VMO and VL of the dominant limb during five repetitions of a step down activity and five repetitions of a straight leg raise exercise. The average intensity of the rectified and smoothed EMG activity from each activity was normalised to that elicited in a maximal quadriceps setting exercise. The ratio of normalised VMO:VL EMG intensity levels was calculated. Results: The median difference in the VMO:VL ratio between the groups was 0.11 [approximate 95% confidence interval (CI) -0.62 to 1.00] during step down and -0.07 (approximate 95% CI -0.26 to 0.20) during straight leg raise. Using Mann Whitney U-tests, these differences were not statistically significant (P = 0.648 and 0.619, respectively). Conclusions: This study found no gender difference in the VMO:VL EMG intensity ratio in asymptomatic participants. This suggests that the difference in incidence of patellofemoral pain syndrome between genders is not influenced by quadriceps intensity ratios, when participants are asymptomatic.