Objective: The prominent influence of skeletal muscle mitochondria on health and physical capacity can be enhanced through diet and exercise. Ketogenic diets have great potential to drive this enhancement, but prior research is limited, particularly in humans. Therefore, the objective of the present research was to characterize changes in skeletal muscle mitochondria function induced by a ketogenic diet during adaptation to chronic exercise. Methods: Twenty-nine participants completed a 12-week supervised exercise program while following a ketogenic diet (KD, n=15, males=13) or their habitual mixed diet (MD, n=14, males=12). Body composition was measured using dual-energy x-ray absorptiometry (iDXA, GE Healthcare, Chicago, IL). Blood was drawn in a fasted and resting state and serum insulin and glucose were measured using an enzyme-linked immunosorbent assay kit (Calbiotech, El Cajon, CA) and a hexokinase reagent set (Pointe Scientific, Canton, MI), respectively. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated based on insulin and glucose values. Respiratory quotient (RQ) was measured through gas exchange (TruOne 2400, Parvo Medics, Sandy, UT). Muscle biopsies were collected from the Vastus lateralis, from which mitochondria were isolated. O2 consumption and membrane potential were measured with a Clark-type electrode fitted with a tetraphenylphosphonium electrode. H2O2 and ATP production were measured using fluorescence (Amplex Ultra Red) and luminescence (luciferase) assays, respectively. Each test was repeated with a carbohydrate- (pyruvate), fat- (palmitoyl-L-carnitine), and ketone-based (β-hydroxybutyrate+acetoacetate) substrate. Results: Participants were matched by age, gender, and body fat (KD vs MD: 27.4±1.8 vs 24.6±2.4 yrs, 25.6±1.3% vs 22.0±2.3%). At baseline, HOMA-IR was greater in KD (2.1±0.3 vs 1.5±0.2, p=0.056) and decreased during the intervention (2.11±0.3 to 1.11±0.1, p=0.008). Weight loss was greater for KD (-6.9±0.9 vs 0.7±0.4 kg, p<0.001), as was decrease in body fat percentage (-5.4±0.7 vs -0.7±0.5 %, p<0.001). Mean daily blood β-hydroxybutyrate concentration for KD was 1.2±0.2 mM and RQ decreased only in KD (0.82 to 0.75, p=0.001), all indicating the ketogenic diet induced a profound shift in energy metabolism towards reliance on fat oxidation. An effect of time was observed for increases in mitochondrial protein (p=0.019) and respiratory control ratio (RCR, p=0.003). Time by diet interactions indicate a lesser increase in H2O2 (p=0.098) and a relative increase in ATP production (p=0.003) and efficiency (based on ATP/O2 and ATP/H2O2, p<0.005) for KD. With the fat-based substrate, RCR and ATP production increased only for KD (4.7±0.3 to 5.6±0.2, p=0.009; 20.9±4.2 to 28.4±4.6 nmol/mg/min, p=0.028). ATP production with the ketone-based substrate was 4 to 8 times lower than with other substrates, indicating that ketones are minimally oxidized in human skeletal muscle. Conclusions: While the effects of time for mitochondrial protein and RCR indicate exercise-induced enhancement of mitochondria function, the time by diet interactions for ATP, ATP/O2, and ATP/H2O2 indicate augmentation of this enhancement by the ketogenic diet, particularly in relation to fat oxidation. The improvement in HOMA-IR for KD suggests that these improvements may have partly been related to rescue of metabolic impairment. Further research is strongly encouraged for determination of mitochondria function as a target through which ketogenic diets improve metabolic health.