PURPOSE: Mitochondrial dysfunction is associated with many conditions including muscle atrophy, dystrophy, and obesity-induced insulin resistance. Increases in mitochondrial-derived reactive oxygen species have been attributed to some of these adverse conditions. Emerging evidence suggests that mechanical stimulation may affect mitochondrial homeostasis and adaptation. However, the direct effect of isolated mechanical stimulation on mitochondrial adaptation in human skeletal muscle needs to be determined. Therefore, this study investigated the effect of percussive massage as a practical form of mechanical stimulation on skeletal muscle mitochondrial respiration and H2O2 production. METHODS: 10 healthy young men and women (22 ± 4 yr) received 18 sessions of 30-min-percussive massage on their right thigh over the course of 6 weeks. Subcutaneous fat was imaged and measured using ultrasound on both the treated and contralateral thighs. Muscle samples were collected one week before the start of massage sessions and 48 h after the last session from the participants treated vastus lateralis. Permeabilized muscle fibers were assessed for maximal respiratory capacity, fat oxidative capacity, and H2O2 flux using high-resolution respirometry (Oroboros O2k). RESULTS Percussive massage treatment significantly increased maximal mitochondrial fat oxidation supported by octanoylcarnitine, malate, and ADP from 13.16 ± 4.9 pmolO2/(s*mg) to 17.33 ± 3.59 pmolO2/(s*mg) (p = 0.032). In accordance with this finding, subcutaneous fat thickness of the treated thigh decreased by 7.3% ± 5.1, which was significantly higher than the contralateral thigh (0.8% ± 5.2; p = 0.004). H2O2 production, was reduced following massage treatment in the presence of glutamate, malate, succinate, and 12.5 μM, 25 μM, 100 μM and 2.5 mM ADP (48.87% ± 29.58, p = 0.034; 54.15% ± 29.33, p = 0.035; 71.76% ± 13.2, p = 0.006; 59.76% ± 53.22 p = 0.011, respectively). However, coupled respiration supported by glutamate, malate, and ADP remained unchanged after 6 weeks of massage treatment (p = 0.8) CONCLUSION: 6 weeks of percussive massage improves mitochondrial fat oxidation and decreases subcutaneous fat thickness and maximal H2O2 production but does not change maximal coupled respiration.