Background: Bed rest represents an extreme model of physical inactivity and leads to age-related changes in cardiovascular and metabolic function. In particular, reduced skeletal muscle mitochondrial capacity and intrinsic mitochondrial function in response to physical inactivity may have broad implications for human disease. The aim of this study was to determine if 21-days of bed test decreased total and intrinsic mitochondrial respiration and if the changes could be mitigated by exercise sessions performed during bed rest. Methods: Subjects (n=9) completed 21-days bed rest without (CON) and with resistive vibration exercise (RVE) using a randomized crossover design. Fat mass was maintained by adjusting dietary intake based on body composition and resting metabolic rate measurements. The physiological response to inactivity was measured by VO2 max, a hyperinsulinemic euglycemic clamp, resting energy expenditure and body composition. The O2 flux capacity of saponin permeabilized skeletal muscle fibres from the vastus lateralis was measured using a carbohydrate and lipid substrate-uncoupler-inhibitor-titration (SUIT) protocol. Respirometry data was normalized to wet weight and citrate synthase activity. Mitochondrial protein content was determined by Western blot analysis. Results: There was a significant reduction in body mass and lean tissue mass (p<0.05) but no change in fat mass following the CON and RVE trials. Insulin sensitivity and VO2max, relative to fat free mass, decreased significantly in the CON but not RVE group (p<0.05). There was a significant reduction in LEAK, OXPHOS and ETS capacity, normalized to wet weight, in the CON but not RVE group (p<0.05). Skeletal muscle citrate synthase activity was significantly lower in both groups (p<0.05) and when used to normalize the respiratory data, only LEAK respiration remained significantly reduced. The change in LEAK significantly correlated with the change in VO2max (p<0.05) in the control group. Mitochondrial proteins representative of complex III and IV are also reduced in both groups. Conclusion: Bed rest is associated with a decrease in skeletal muscle mass, insulin sensitivity and whole body oxygen consumption, independent of fat mass. Many of these changes can be prevented by resistance vibration exercise. The decrease in OXPHOS and ETS were not significantly different when normalised to citrate synthase suggesting the changes could be related to mitochondrial content or function. However, the change in LEAK respiration was independent of citrate synthase and exercise training. This may be due to reduced uncoupling of substrate oxidation and ATP synthesis and is supported by a reduction in the coupling control ratio. In conclusion, a decrease in LEAK respiration is one of the earliest changes associated with physical inactivity and may be important for long term changes in metabolic function.