The maintenance of muscle function is extremely important for whole body health and exercise is essential to this process. The ubiquitin-proteasome system (UPS) is required for muscle adaptation following exercise but little is known about acute post-translational regulation and proteome remodelling during exercise. Here, we used quantitative proteomics to study ubiquitin-signalling dynamics in human skeletal muscle biopsies from healthy males before, during and after a single bout of high-intensity exercise. Exercise resulted in a marked depletion of protein ubiquitylation consistent with proteasome activation. This was also associated with acute post-translational modification of protein abundance via a network of proteases.