Mitochondrial function has previously been studied in ageing, but never in humans matched for maximal oxygen uptake (). Furthermore, the influence of ageing on mitochondrial substrate sensitivity is not known.

Skeletal muscle mitochondrial respiratory capacity and mitochondrial substrate sensitivity were measured by respirometry in young (23 ± 3 years) and middle‐aged (53 ± 3 years) male subjects with similar . Protocols for respirometry included titration of substrates for complex I (glutamate), complex II (succinate) and both (octanoyl carnitine) for calculation of substrate sensitivity (C₅₀). Myosin heavy chain (MHC) isoforms, citrate synthase (CS) and β‐hydroxy‐acyl‐CoA‐dehydrogenase (HAD) activity, mitochondrial DNA (mtDNA) content, protein levels of complexes I–V and antioxidant defence system [manganese superoxide dismutase (MnSOD)] were measured.

No differences were found in maximal mitochondrial respiration or C₅₀ with glutamate (2.0 ± 0.3 and 1.8 ± 0.3 mm), succinate (3.7 ± 0.2 and 3.8 ± 0.4 mm) or octanoyl carnitine (47 ± 8 and 56 ± 7 μm) in young and middle‐aged subjects respectively. Normalizing mitochondrial respiration to mtDNA young subjects had a higher (P < 0.05) respiratory capacity per mitochondrion compared to middle‐aged subjects. HAD activity and mtDNA per mg tissue were higher in middle‐aged compared to young subjects. Middle‐aged had a higher MHC I isoform and a lower MHC IIX isoform content compared to young subjects.

Mitochondrial substrate sensitivity is not affected by ageing. When young and middle‐aged men are carefully matched for , mitochondrial respiratory capacity is also similar. However, per mitochondrion respiratory capacity was lower in middle‐aged compared to young subjects. Thus, when matched for , middle‐aged seem to require a higher mitochondrial content than young subjects.