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High-intensity interval training (HIT) increases skeletal muscle oxidative capacity similar to traditional endurance training, despite a low total exercise volume. Much of this work has focused on young active individuals, and it is unclear whether the results are applicable to older less active populations. In addition, many studies have used "all-out" variable-load exercise interventions (e.g., repeated Wingate tests) that may not be practical for all individuals. We therefore examined the effect of a more practical low-volume submaximal constant-load HIT protocol on skeletal muscle oxidative capacity and insulin sensitivity in middle-aged adults, who may be at a higher risk for inactivity-related disorders.Seven sedentary but otherwise healthy individuals (three women) with a mean ± SD age, body mass index, and peak oxygen uptake (V˙O2peak) of 45 ± 5 yr, 27 ± 5 kg·m−2, and 30 ± 3 mL·kg−1·min-1 performed six training sessions during 2 wk. Each session involved 10 × 1-min cycling at ∼60% of peak power achieved during a ramp V˙O2peak test (eliciting ∼80%-95% of HR reserve) with 1 min of recovery between intervals. Needle biopsy samples (vastus lateralis) were obtained before training and ∼72 h after the final training session.Muscle oxidative capacity, as reflected by the protein content of citrate synthase and cytochrome c oxidase subunit IV, increased by ∼35% after training. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α was increased by ∼56% after training, but the transcriptional corepressor receptor-interacting protein 140 remained unchanged. Glucose transporter protein content increased ∼260%, and insulin sensitivity, on the basis of the insulin sensitivity index homeostasis model assessment, improved by ∼35% after training.Constant-load low-volume HIT may be a practical time-efficient strategy to induce metabolic adaptations that reduce the risk for inactivity-related disorders in previously sedentary middle-aged adults.