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Ageing is associated with significant impairments in musculoskeletal and metabolic health, predisposing older adults to gradual loss of physical function. Evidence for the detrimental impact of physical inactivity and sedentary behaviour on health is emerging. Previous experimental protocols have involved overt forms of physical inactivity, with little relevance to the healthy, ageing population. Step-reduction (SR) interventions provide a useful, valid research design for investigating physical inactivity in free-living participants. The influence of ageing on the musculoskeletal and metabolic response to these periods of physical inactivity with increases in sedentary behaviour is unknown. 26 young (28±8y) and 21 older (60±6y) habitually active (>10,000 steps per day) participants were recruited. Dual-energy x-ray absorptiometry (DEXA), magnetic resonance spectroscopy (MRS), bone turnover biochemistry, a cardiorespiratory fitness test, a peak power leg press and an oral glucose tolerance test (OGTT) were performed. These measures were performed at baseline, following 14 days of step reduction (~1,500 steps per day) and after 14 days of returning to habitual activity. Independent t-tests and analysis of covariance (ANCOVA) models were utilised; baseline data is presented as mean difference between groups  SD whereas, intervention data is presented as mean difference between time points (intervention effect) and between age groups (interaction effect) plus 95%CI. Statistical significance was delimited at 0.05. Step count and PA behaviour were similar between young and older groups at baseline, equally; time spent being sedentary was comparable between age groups (P > 0.05). During SR, step count reduced by 10752±2943 steps/day in the young (83±7%) and 10204±2338 steps/day in the older group (84±7%), with concomitant increases in sedentary time compared with baseline (P < 0.001). SR induced significant losses in total body lean mass (-0.26 [95%CI -0.49, 0.02kg]; P<0.001) and lean leg mass (-0.10 [95%CI -0.02, -0.04kg]; P < 0.001) accompanied by significant gains in total body (0.31 [95%CI 0.19, 0.83kg]; P=0.01) and abdominal fat (0.76 [95%CI 0.25, 1.3kg]; P=0.003). All of these changes were similar between age groups (P > 0.05). IHCL (0.85 [95%CI 0.16, 1.54%]; P < 0.001) and IMAT (1.83 [95%CI 0.71, 2.95%]; P < 0.001) significantly increased during SR, but the effect was greater in the older group (P < 0.05). Decreases in maximum leg strength (-0.34 [95%CI -0.52, -0.15kg-1]; P < 0.001) and peak power (-0.77 [95%CI -0.70, 0. 54w.kg-1]; P < 0.001) relative to lean mass were evident. Trends suggest that reductions tended to be greater in the older group (P < 0.10). There were no changes in gait speed, but handgrip strength declined during SR (-1 [95%CI -2, 0kg]; P=0.03), with no difference between age groups (P=0.22). CRF (-2.65 [95%CI -3.80, -1.50ml.min.kg-1]; P < 0.001) and mitochondrial function (rate constant: -0.26 [95%CI -0.43, -0.08ks-1]; P=0.002) declined during SR. These reductions were greater in older adults (P=0.04). Whole-body IS decreased (-0.74 [95%CI -1.02, -0.45]; P < 0.001) and skeletal muscle IR increased (0.78 [95%CI 0.14, 1.41]; P < 0.01) during SR, with no difference between groups (P > 0.05). Equally, serum cholesterol (0.27 [95%CI 5, 0.04], 0.49 mmol.l; P=0.02) and TG (0.32 [95%CI 0.15, 0.50mmol.l]; P < 0.001) increased in both groups to similar extents (P > 0.05). BMD significantly declined during SR (-0.01, 95% CI -0.01, 0.00; P=0.45) similarly in both groups (P > 0.05), however, there was no significant change in bone turnover markers (P > 0.05). PA is effective in preserving metabolic health and mitochondrial function during ageing. Even short-term reductions induce substantial musculoskeletal and metabolic declines, which were greater in the older group compared with the young. These novel data suggest that older adults are more at risk of the detrimental effects of physical inactivity. These findings highlight the importance of maintaining PA throughout the lifespan.