Purpose: This investigation evaluated the effects of a high-load (50% body weight) eccentric exercise training protocol on reloading myofibrillar damage in soleus (SOL) and extensor digitorum longus (EDL) muscles in rats following 7 days (d) of hindlimb suspension unloading (HSU). Methods: 48 female Sprague-Dawley rats were randomly stratified to four experimental groups; exercise + hindlimb suspension unloading (ExHSU), hindlimb suspension unloading (HSU), exercise (Ex) and control (C). The ExHSU and Ex groups underwent a high-load eccentric exercise protocol for ~2.5 weeks. Following exercise training, the ExHSU and HSU groups underwent 7 d of hindlimb suspension unloading and a subsequent 16-19 h reloading period. ANOVA was used to determine significance between groups for the following variables: body weight (BW) across time, BW at sacrifice, Glucose-6-phosphate dehydrogenase (G-6-PDH) activity, fiber area, fiber area to body-weight ratio, % myofibrillar damage, SOL and EDL wet, dry and wet-weight to body-weight ratios, % interstitial area, adrenal weights and adrenal weight to body-weight ratios, tibia lengths and tibia bone mineral content. Results: ANOVA revealed no significant differences (p > .10) between the ExHSU and HSU groups for BW at sacrifice, fiber area, fiber area to body-weight ratio, SOL and EDL wet, dry and wet-weight to body-weight ratios, adrenal weights and adrenal weight to body-weight ratios and tibia lengths and bone mineral content. Yet a post analysis t-test revealed a significantly higher % of myofibrillar damage in the HSU vs. the ExHSU group. Further, G-6-PDH activity and % interstitial area approached significance (p = 0.134 and p = 0.152, respectively). Conclusions: The high-load eccentric exercise training protocol prior to HSU attenuated the % of myofibrillar damage during reloading. Further, the % of interstitial area and G-6-PDH activity tended to be smaller in the ExHSU group vs. the HSU group. Therefore, eccentric exercise prior to HSU may elicit a repeated bout effect and attenuate the amount damage incurred by the muscle during reloading. Additionally, this investigation was the first to demonstrate increased G-6-PDH activity with reloading myofibrillar damage.