Skeletal muscle is a plastic tissue that adapts to exercise in many ways. Differential proteome profiling can be used to obtain a global view of proteins that change expression following a repeat bout of eccentrically‐biased exercise, as well as its gender specificity. Exercise‐naive male and female mice (C57BL/10ScSnJ 000476 +/+) ran downhill twice one week apart (‐15°) on a treadmill at 25 m s−1 for 15 min and 800 ug of total biceps brachii extract was electrophoresed on pH 5‐8 2‐D gels. The resulting spots that changed at least +/‐ 2‐fold and were statistically significantly different (p<0.05) relative to the unexercised controls using PDQuest 8.0 were analyzed by LC/MS‐MS and identified using BioWorks 3.3.1. Significant proteome remodeling occurs subsequent to the repeat bout. The expression change patterns were followed at 0, 24, 48, 72 and 168 hrs post‐repeat bout; significant changes were observed in myofibrillar and cytoskeletal proteins, creatine kinase and many of the glycolytic enzymes. The cellular stress responses were unique to individual heat shock and oxidative stress proteins. More protein spots changed in females (299) than in males (236); the female changes peaked at 24 hr post‐exercise, whereas the male changes did not show much variation over the week following the repeat bout. Supported by NSF 0420971, the Smith College Blakeslee Fund and HHMI.