["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend Three weeks of overtraining in trained endurance athletes reduced exercise performance without impairing glucose tolerance. In skeletal muscle, overtraining was associated with increased expression of lipid metabolism‐related proteins, enhanced mitochondrial biogenesis and preserved insulin signalling, despite elevated oxidative stress. Combined, these data indicate that excessive training does not induce mitochondrial dysfunction or insulin resistance in skeletal muscle.\n\n\n\n\n\n\n\n\n\nAbstract\nExcessive training, also known as overtraining, has been suggested to impair skeletal muscle mitochondrial function and glucose homeostasis, challenging the notion that exercise is inherently beneficial. However, methodological limitations on assessment of mitochondrial bioenergetics while considering exercise‐induced mitochondrial biogenesis make the metabolic consequences of overtraining still debatable. Therefore, we investigated skeletal muscle insulin signalling and mitochondrial bioenergetics in skeletal muscle following a 3‐week overtraining protocol in healthy highly trained endurance athletes. Proteomics of skeletal muscle revealed an upregulation of proteins related to fatty acid metabolism and mitochondrial content induced by overload training. Functionally, mitochondrial respiratory capacity as well as H2O2 emission were increased in permeabilized muscle fibres. These effects were dependent on mitochondrial content, suggesting preservation of intrinsic mitochondrial oxidative phosphorylation. While sub‐maximal mitochondrial H2O2 emission and oxidative stress were increased following excessive training, insulin signalling within skeletal muscle (i.e., Akt phosphorylation) during an oral glucose challenge was improved, suggesting excessive exercise does not induce skeletal muscle insulin resistance. In a further analysis, based on their psycho‐physiological performances, participants were identified by who successfully or not developed an overreaching phenotype. This approach revealed a unique proteome signature in individuals who were overreached, marked by a smaller increase in proteins involved in cytoskeleton organization, glycogen metabolism, and protein translation. However, despite such classification, we did not observe reductions in either mitochondrial bioenergetics or insulin signalling within skeletal muscle. Altogether, overtraining in highly active individuals induces mitochondrial biogenesis without impairments in skeletal muscle insulin signalling nor mitochondrial oxidative capacity.\n\n\n\n\n\n\n\n\n\nKey points\n\nProteomics revealed upregulation of fatty acid and mitochondrial proteins by excessive training.\nOvertraining does not cause mitochondrial dysfunction.\nImproved insulin signalling in skeletal muscle post‐overtraining.\nOverreached athletes show blunted increase in protein synthesis and metabolism.\n\n\n"]