The objective of this study was to test the hypothesis that muscle disuse can result in abnormal neuromuscular transmission. Six healthy volunteers (2 females, mean age = 33 years) participated in the study. Cast immobilization of one leg, for a period of 4 weeks, was used as a model of disuse; the contralateral leg was used as a control. Three testing sessions, consisting of stimulation single fiber electromyography of the soleus muscle bilaterally, were done before, immediately after, and 4 weeks after cast removal. The main outcome measure was neuromuscular jitter, measured as the mean of the consecutive differences (MCD) of single muscle fiber potentials. A mean MCD was calculated for each muscle, and the data from all subjects was pooled to calculate a sample mean MCD. The control side showed good reproducibility of results between testing sessions. The experimental side showed a significant increase of the sample mean MCD after casting when compared to: preimmobilization values (P < 0.001), and to the control side (P < 0.001). After 4 weeks of recovery, the sample mean MCD returned to preimmobilization values. This study presents evidence that muscle disuse, without any neuromuscular disease, can result in a reversible dysfunction of neuromuscular transmission.