Ultra-endurance exercise is associated with stresses that mechanically damage muscle cells and lead to injured proteins and organelles. Previous investigations provided evidence that ultra-endurance exercise is associated with a coordinated up-regulation of the ubiquitin-proteasome and autophagy-lysosomal proteolytic pathways (Jamart et al., 2012). The purpose of the present investigation was to assess the resulting modifications in the muscle proteome. Ten men, experienced ultra-endurance athletes ran for 24h on a treadmill. Muscle biopsy samples were taken from the vastus lateralis muscle 2h before starting and immediately after finishing exercise. Athletes ran 150+16 km with an effective running time of 18h:42min (±41min). Label-free quantitative protein profiling (‘Shot-Gun’) was performed (Théron et al., 2014) to quantify and compare proteomes before and after ultra-endurance running. Shot-Gun proteomics of the Human muscle homogenate identified 633 proteins, and among them 96 were differentially expressed after ultra-endurance running. Most of the proteins were under-represented after exercise. Functional interaction networks indicated that ultra-endurance strongly altered the mitochondrial proteome suggesting enhanced mitophagy. Our results also revealed important modifications related to the cytoskeleton, cytodetoxification, proteostasis and membrane repair. This study describes the most extensive proteomic analysis of Human muscle adaptation to ultra-endurance exercise. Many potential biomarkers may represent novel starting points to elucidate the mechanisms of muscle adaptation to extreme exercise