Ageing affects mitochondrial integrity in skeletal muscle, and physical inactivity may further exacerbate these changes. Although mitochondrial alterations are documented in ageing and disuse independently, how disuse impacts the mitochondrial phenotype in older populations remains unclear. This work aimed to characterise how physical inactivity impacts mitochondrial function, morphology and gene expression in the skeletal muscle of older adults. Ten healthy older men (65+ years) underwent 10 days of bed rest. Skeletal muscle biopsies were collected before and after bed rest to assess mitochondrial respiration (high-resolution respirometry), H2O2 emission, mitochondrial protein expression, morphology and volume density (electron microscopy) and transcriptomic profile. Ten days of inactivity increased mitochondrial reactive oxygen species (ROS) emission under non-phosphorylating conditions but did not impair oxidative phosphorylation (OXPHOS) capacity, indicating preserved respiratory efficiency. Consistently, mitochondrial respiratory complex and supercomplex protein abundance were unchanged. Mitochondrial mass decreased, as shown by reduced mitochondrial volume density. Reduced dynamin-like protein 1 (DRP1) phosphorylation at serine 637 was observed, whereas other mitochondrial fission and fusion protein levels remained unchanged. Mitochondrial morphology remained unaltered. Transcriptomic analysis revealed >3000 differentially expressed genes, characterised by downregulation of oxidative phosphorylation genes alongside altered mitophagy, antioxidant and oxidoreductase pathways. In summary, 10-day bed rest increased mitochondrial ROS emission and reduced mitochondrial mass in older skeletal muscle despite preserved respiratory function, indicating that elevated ROS production occurs upstream of respiratory dysfunction and is potentially linked to impaired antioxidant defence and ROS clearance. These findings suggest that preserving redox balance during inactivity may be a key strategy to maintain muscle health and functional independence in ageing populations. KEY POINTS: The impact of short-term physical inactivity on mitochondrial function within the context of ageing remains poorly defined. This study examined the impact of 10-day bed rest on skeletal muscle mitochondrial function, morphology and gene expression in older adults. Short-term inactivity increased mitochondrial ROS production, accompanied by a dysregulation of antioxidant and oxidoreductase genes, indicating a reduced capacity for ROS clearance. Mitochondrial respiration was preserved under both submaximal and maximal stimulation. When normalised to mitochondrial content (citrate synthase activity), respiratory capacity increased, suggesting improved intrinsic efficiency. Mitochondrial mass was reduced, supported by decreased mitochondrial volume density assessed morphologically. Transcriptomic alterations in the mitophagy pathway suggest a potential role of altered mitochondrial degradation in this reduction. These findings indicate a transient compensatory response of ageing mitochondria to short-term disuse, suggesting that functional impairments are likely driven by cardiovascular and microvascular factors rather than mitochondrial respiration itself.
