Type 2 diabetes (T2D) is associated with excess energy intake and physical inactivity. Intrinsically linked to these events are deficits in muscle total mitochondrial volume and/or function (Kelley et al., 2002), but whether this reflects a lower muscle mitochondrial DNA (mtDNA) copy number or simply a muscle deconditioning is debated. We therefore quantified maximal oxygen consumption (VO2max), mtDNA copy number (RQ-PCR) and mitochondrial enzyme activities (GluDH, CS, β-HAD; spectrophotometrically) in muscle biopsies from older age-matched, male volunteers: healthy trained (57.4 ± 0.9 yrs, n=10), healthy sedentary (60.0 ± 2.0 yrs, n=10), and T2D patients (58.9 ± 2.0 yrs, n=10). Ethical approval was granted before subject’s consent was obtained. VO2maxa ISIb mtDNA GluDHc CSc β-HADc Trained 3.8±0.1*# 133±24** 1461±52***### 9.8±0.9**# 84.4±9.5**# 32.6±3.2*# Sedentary 3.2±0.2 86±12* 749±35*** 6.9±0.4 51.9±7.0 21.3±2.5 T2D 2.9±0.2 48±6 454±59 5.6±0.6 42.6±2.3 20.9±3.6 aL min-1; bmg L2 (mmol mU min)-1; cmmol min-1mg-1 protein, *, **, ***Significantly different from T2D (P<0.05, P<0.01 and P<0.001); #, ###Significantly different from sedentary (P<0.05, P<0.001). All values in text and Table represent mean±SEM. Statistical differences detected using ANOVA. Except for Insulin Sensitivity Index (ISI), no differences in mitochondrial enzymatic markers were detected between older healthy sedentary and T2D (Table). However, both were markedly different from older trained volunteers. The number of mtDNA copy number in the older trained group was significantly greater than in the older untrained and T2D groups (P<0.001). Furthermore, the mtDNA copy number in the older untrained group was significantly greater than in the T2D group (P<0.001). This suggests that any apparent muscle mitochondrial dysfunction in T2D likely results from a decline in total mtDNA copy number.