The purpose of this study was to determine the measurement error associated with antagonist muscle activity in isometric knee strength testing at 60 degrees of knee flexion in both sexes. Muscle specific EMG-contraction intensity relationships were obtained from 22 young people by having them match moment targets ranging from 10% to 100% peak moment. The moments attributed to each of the quadriceps and hamstrings muscles were partitioned using a practical mathematical model. Subject specific EMG-moment relationships were defined for each muscle using second-order polynomial equations. These equations were subsequently used to predict the countermoment associated with antagonist muscle activity. Error during strength testing was calculated by expressing net antagonist moments as a percentage of net agonist moments. The net antagonist moments associated with quadriceps and hamstrings muscle activity were 11.0% and 8.7% of the peak moment values recorded when the same muscle groups were acting as agonists. The error associated with antagonist activity was significantly higher in knee flexion (20.1%) than in knee extension (4.5%). Females displayed significantly higher error in knee flexor testing (P < 0.001). Limb symmetry indices did not change significantly when the countermoments generated by the antagonist muscles were accounted for (P > 0.05). The results of this study indicate that the error associated with antagonist activity in knee extensor testing is relatively small, whereas the error in knee flexor testing is larger. This is due to the quadriceps being much stronger than the hamstrings muscles while displaying similar levels of antagonist activity.