A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by ∼2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.