Exercise training induces mitochondrial biogenesis, while high‐fat diet increases the ability of mitochondria to use long and short‐chain fatty acids

) and ∼14% of LCFA (palmitoylcarnitine)-linked respiration. Despite this low capacity, the intrinsic mitochondrial ability to oxidize palmitoylcarnitine, acetate and butyrate increased (P < 0.05: +50%) following HFD consumption, suggesting HFD rewires mitochondria to optimize lipid oxidation. By contrast, exercise training prevented these HFD-induced intrinsic mitochondrial responses. Although intrinsic changes are biologically relevant, skeletal muscle adaptation to metabolic stress also involves mitochondrial biogenesis and an expansion of the mitochondrial proteome. Proteomic analysis and citrate synthase activity revealed that, although HFD independently did not alter mitochondrial protein abundance, exercise training increased mitochondrial proteins, a response amplified in the presence of a HFD. Consequently, although exercise did not directly enhance mitochondrial SCFA-supported respiration, the combined effect of HFD and exercise predicted a greater overall capacity for SCFA oxidation because of increased mitochondrial abundance. Collectively, although SCFAs contribute minimally to mitochondrial respiration in skeletal muscle, combined HFD and exercise synergistically enhance overall OXPHOS capacity across diverse substrates, including SCFAs, primarily through increased mitochondrial protein abundance rather than intrinsic mitochondrial remodelling. KEY POINTS: Peroxisome and gut derived short-chain fatty acids (SCFA) have been proposed as an alternative metabolic fuel source to support skeletal muscle oxidative phosphorylation. The capacity and adaptability of mitochondrial SCFA oxidation remains unknown. SCFA-supported mitochondrial respiration is limited (<15%) compared to carbohydrate (pyruvate) and long-chain fatty acid linked substrates. High-fat feeding increased the intrinsic capacity of mitochondria to utilize palmitoylcarnitine, acetate and butyrate- effects prevented by 4 weeks of exercise training. Combined high-fat diet and exercise training increased skeletal muscle mitochondrial protein content in an additive manner, increasing oxidative capacity and ability to utilize both long- and SCFAs as a fuel source.