Although the role of satellite cells has been confirmed during skeletal muscle growth and regeneration, their involvement during work-induced muscle growth remains uncertain. In this study, chronically overloaded rat soleus muscles were ultrastructurally monitored following surgical ablation of synergists to examine cytological adaptations of satellite cells and myofibers. The left soleus muscle of 20 female Spague-Dawley rats (7 weeks of age) was induced to hypertrophy by excising the contralateral plantaris and gastrocnemius muscles under pentobarbital anesthesia. Right limbs were sham-operated and served as controls. On days 3, 7, 14, 21, and 30 after surgery, the soleus muscles were removed and processed for electron microscopy. Two morphologically distinct phases were noted in the surgically overloaded muscles. The first stage (week 1) was characterized by a significant increase in the number of satellite cells, and by more than half of the experimental muscle fibers displaying myofibrillar disruptions, mitochondrial alterations and glycogen pooling. The second phase (weeks 2–4) featured mostly normal, although larger appearing muscle fibers, with the satellite cell frequency remaining slightly elevated. These findings suggest that muscle fiber structural abnormalities, rather than an increase in muscle activity, may play a more significant role in the early activation of satellite cells during compensatory hypertrophy, whereas activation of satellite cells during the later stages may be in response to increased levels of muscle activity.