Recent studies have reported that endurance exercise enhances mitochondrial function, facilitating discussions of its potential as a therapeutic strategy for mitochondrial diseases caused by the accumulation of mutant mitochondrial DNA (mtDNA). In this study, we assessed the effects of endurance exercise on muscle pathology in a mitochondrial disease mouse model (mito-miceΔ) that is characterized by severe clinical phenotypes owing to the predominant accumulation of mtDNA with a large-scale deletion (ΔmtDNA). Contrary to expectations that endurance exercise may enhance mitochondrial function, endurance exercise exacerbated muscle pathology in mito-miceΔ. Therefore, exercise interventions should be potentially avoided in patients with severe mitochondrial diseases.