KEY POINTS:A classic unresolved issue in human integrative physiology involves the role of exercise intensity, duration and volume in regulating skeletal muscle adaptations to training. We employed counterweighted single-leg cycling as a unique within-subject model to investigate the role of exercise intensity in promoting training-induced increases in skeletal muscle mitochondrial content. Six sessions of high-intensity interval training performed over 2 weeks elicited greater increases in citrate synthase maximal activity and mitochondrial respiration compared to moderate-intensity continuous training matched for total work and session duration. These data suggest that exercise intensity, and/or the pattern of contraction, is an important determinant of exercise-induced skeletal muscle remodelling in humans. ABSTRACT:We employed counterweighted single-leg cycling as a unique model to investigate the role of exercise intensity in human skeletal muscle remodelling. Ten young active men performed unilateral graded-exercise tests to measure single-leg V̇O2, peak and peak power (Wpeak ). Each leg was randomly assigned to complete six sessions of high-intensity interval training (HIIT) [4 × (5 min at 65% Wpeak and 2.5 min at 20% Wpeak )] or moderate-intensity continuous training (MICT) (30 min at 50% Wpeak ), which were performed 10 min apart on each day, in an alternating order. The work performed per session was matched for MICT (143 ± 8.4 kJ) and HIIT (144 ± 8.5 kJ, P > 0.05). Post-training, citrate synthase (CS) maximal activity (10.2 ± 0.8 vs. 8.4 ± 0.9 mmol kg protein-1 min-1 ) and mass-specific [pmol O2 •(s•mg wet weight)-1 ] oxidative phosphorylation capacities (complex I: 23.4 ± 3.2 vs. 17.1 ± 2.8; complexes I and II: 58.2 ± 7.5 vs. 42.2 ± 5.3) were greater in HIIT relative to MICT (interaction effects, P < 0.05); however, mitochondrial function [i.e. pmol O2 •(s•CS maximal activity)-1 ] measured under various conditions was unaffected by training (P > 0.05). In whole muscle, the protein content of COXIV (24%), NDUFA9 (11%) and mitofusin 2 (MFN2) (16%) increased similarly across groups (training effects, P < 0.05). Cytochrome c oxidase subunit IV (COXIV) and NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9) were more abundant in type I than type II fibres (P < 0.05) but training did not increase the content of COXIV, NDUFA9 or MFN2 in either fibre type (P > 0.05). Single-leg V̇O2, peak was also unaffected by training (P > 0.05). In summary, single-leg cycling performed in an interval compared to a continuous manner elicited superior mitochondrial adaptations in human skeletal muscle despite equal total work.