Significance Mitochondria are major cellular energy suppliers and have to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. We obtained the first, to our knowledge, comprehensive mitochondrial proteome around the clock and identified extensive oscillations in mitochondrial protein abundance that predominantly peak during the early light phase. Remarkably, several rate-limiting mitochondrial enzymes that process different nutrients accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. Concurrently, we uncovered daily oscillations in mitochondrial respiration that are substrate-specific and peak during different times of the day. We propose that the circadian clock PERIOD proteins regulate the diurnal utilization of different nutrients by the mitochondria and thus, optimize mitochondrial function to daily changes in energy supply/demand.