The purpose of this research was to provide more definitive support for the hypothesis that prolonged muscle activation at high intensities increases voluntary activation deficits. Interpolated twitch responses were evoked during maximal and sub-maximal voluntary contractions of the soleus muscle in 10 college-aged students. Maximal voluntary contractions (MVC), maximal muscle twitches, and interpolated twitch responses were measured before, during, and after fatiguing isometric exercise, five bouts of 20 intermittent MVCs. The relationship between voluntary activation and force was studied by evoking interpolated twitches during sub-maximal voluntary contractions on Day 1 and pre-post fatigue on Day 2. Intraclass reliability coefficients for the MVC, maximal muscle twitch, and interpolated twitch responses were adequate across trials and days (R > or = .80). MVC force and maximal twitch force decreased after the fatiguing exercise bouts by 28% and 32%, respectively (p < .05). Voluntary activation of the fatigue-resistant soleus muscle decreased by 10% after the first five min of maximal exercise with a subsequent decrease of 9% occurring after 25 min of maximal exercise (p < .05). At the end of the experimental session, approximately 30 min after the end of the fatiguing exercise, decreases in 100% MVC force, maximal muscle twitch force, and voluntary activation were still evident: 22%, 23%, and 11%, respectively (p < .05). Post-fatigue, there were also changes in neural strategies for voluntary activation of the soleus muscle at the higher sub-maximal efforts, > or = 70% MVC target levels (p < .05). These data demonstrate the cumulative effects of prolonged exercise on voluntary activation.