Hindlimb unloading (HU) results in oxidative stress, skeletal muscle atrophy, and increased damage upon reloading. Heat shock proteins (HSPs) protect against oxidative stress. However, it is unknown whether HSPs are depressed with long-term unloading (28 days) or reloading. We tested the hypotheses that long-term HU would depress Hsp70 and Hsp25 pathways, whereas reloading would allow recovery in the soleus. Adult Sprague-Dawley rats were divided into three groups: controls; HU for 28 days; and HU + 7 days of reloading (HU-R). Soleus mass decreased with HU, and did not recover to control values with reloading. Hsp70 decreased with HU (-78.5%) and did not recover with HU-R (-81.4%). Upstream heat shock factor-1 was depressed with HU and HU-R. Hsp25 was reduced with HU, but recovered with reloading. Downstream of Hsp25, NADP-specific isocitrate dehydrogenase and glutathione peroxidase decreased with unloading, but only NADP-specific isocitrate dehydrogenase recovered with HU-R. Lipid peroxidation increased in both HU and HU-R. These data indicate that prolonged unloading and subsequent reloading results in complex, differential regulation of Hsp70 and Hsp25 pathways in the rat soleus muscle. Thus dysregulation and uncoupling of the Hsp70 and Hsp25 pathways may lead not only to muscle atrophy with prolonged unloading, but also impaired recovery of muscle mass during early reloading.