In two-stroke apparent movement, repeated presentation of a two-frame pattern displacement followed by a brief inter-stimulus interval (ISI) can create an impression of continuous forward motion (G. Mather, 2006). Does the ISI in two-stroke motion just break the connection between adjacent frames, switching off the motion signal they normally generate, or does it actually generate a reversed motion signal? Reversed apparent motion in two-frame stimuli separated by a brief ISI has been reported in several previous papers (ISI reversal), which found that the effect is optimal at short, mean-luminance ISIs, and is abolished at scotopic luminances. A series of five experiments compared two-stroke apparent motion with ISI reversal using the same stimulus display. The two effects show the same dependence on ISI duration and luminance and are both abolished at low mean luminance. Results therefore support the conclusion that the ISI in two-stroke apparent motion does contribute a reversed motion signal and constrain theoretical explanations of two-stroke apparent motion.