Synchronization between single motor unit (SMU) discharges in separate muscles has been estimated from peaks in averaged electromyographic (EMG) recordings from one muscle triggered from SMU discharge in another. This study evaluated the effect of EMG signal cancellation on this measure of synchronization. SMU activity was recorded with 8 fine-wire electrodes in vastus medialis obliquus (VMO) and vastus lateralis (VL) during gentle isometric knee extension in 7 subjects. Data from 5 VL recordings were summed then rectified, or rectified then summed, to produce multi-unit recordings with and without cancellation, respectively. Averages of summed VL data were triggered from VMO SMUs. Synchronization, defined as a peak >3 SD above the triggered average mean, occurred in 73.68% and 78.95% of recordings with and without cancellation, respectively. To further investigate the effect of cancellation on synchronization, 250 "virtual" EMG recordings were created from VL data. VL SMUs were sorted and modified with respect to discharge rate, amplitude and polarity to create a collection of possible SMU discharge patterns. Virtual recordings were added one-by-one to VL recordings that showed synchronization. Virtual channels were rectified then added or added then rectified, to create data with and without cancellation. Identification of synchronization decreased similarly in both conditions with addition of virtual data. Our data show estimation of synchronization from triggered averages is more likely to detect synchronization in recordings with fewer SMUs, but cancellation has little effect. Synchronization must be interpreted with caution if number of SMUs changes between conditions.