Tissue Doppler imaging (TDI) is typically used to image and quantify tissue motion. We investigated whether this method would serve as a viable alternative to surface electromyography (EMG) in providing a reliable and valid measure of the onset of muscle activity. Ten healthy subjects performed maximal knee extension exercises at 0 degrees /s (isometric), 60 degrees /s, 120 degrees /s, 180 degrees /s, and 240 degrees /s (5 times each, on each side), using an isokinetic dynamometer. Simultaneous EMG and TDI velocity (superimposed on motion-mode ultrasound cine-loops) recordings were made from vastus lateralis. All tests were repeated 1 week later. There was a good correlation between the onset times determined with TDI velocity and EMG: r = 0.78 (day 1), and r = 0.80 (day 2) (each P < 0.001). The mean difference (and SD) in muscle onset time between the two methods (TDI minus EMG) was -20.3 +/- 31.0 ms (day 1) and -17.4 +/- 27.2 ms (day 2). TDI represents a reliable and valid measure of detecting onset of muscle activity. The mean difference between EMG and TDI onset times (approximately 20 ms) is likely explained by electromechanical delay. TDI represents a viable method for measuring the onset of muscle activity; it may offer a non-invasive alternative to fine-wire EMG for use with small or deep muscles.