Muscle disuse impairs muscle quality, and is associated with increased mortality. Little is known regarding additive effects of multiple bouts of disuse, a common occurrence in patients experiencing multiple surgeries. Mitochondrial quality is vital to muscle health and quality; however, to date mitochondrial quality control has not been investigated following multiple bouts of disuse. Therefore, the purpose of this study was to investigate mitochondrial quality controllers during multiple bouts of disuse by hindlimb unloading. METHODS: Male rats (n~8/group) were assigned to the following groups: hindlimb unloading for 28 days, hindlimb unloading with 56 days of reloading, 2 bouts of hindlimb unloading separated by a recovery phase of 56 days of reloading, 2 bouts of hindlimb unloading and recovery after each disuse, or control animals with no unloading. At designated time points, tissues were collected for mRNA and protein analysis of mitochondrial quality. RESULTS: Measures of mitochondrial biogenesis, such as PGC-1α, decreased 30-40% with unloading with no differences noted between unloading conditions. Measures of mitochondrial translation, were 40-50% lower in unloading conditions, with no differences noted between bouts of unloading. Measures of mitophagy were 40-50% lower with reloading, with no differences noted between reloading conditions. CONCLUSION: Disuse causes alterations in measures of mitochondrial quality, however, multiple bouts of disuse does not appear to have additive effects. Novelty: • Disuse atrophy causes multiple alterations to mitochondrial quality control. • With sufficient recovery most detriments to mitochondrial quality control are fixed. • In general, multiple bouts of disuse do not produce additive effects.