Several weeks of intense endurance training enhances mitochondrial biogenesis in humans. Whether a single bout of exercise alters skeletal muscle mitochondrial DNA (mtDNA) content remains unexplored. Double-stranded mtDNA, estimated by slot-blot hybridization and Real Time PCR (RT-PCR) and expressed as mtDNA/nuclear DNA ratio (mtDNA/nDNA), was obtained from the vastus lateralis muscle of healthy human subjects to investigate if skeletal muscle mtDNA changes during fatiguing and nonfatiguing prolonged moderate intensity (2.0-2.5 h; ~60% VO₂max) and short repeated high intensity exercise (5-8 min; ~110% VO₂max). In control resting and light exercise (2 h; ~25% VO₂max) studies, mtDNA/nDNA did not change. Conversely, mtDNA/nDNA declined after prolonged fatiguing exercise (0.863±0.061 vs. 1.101±0.067 at baseline; n=14; P=0.005), remained lower after 24 h of recovery and was restored after 1 week. After non-fatiguing prolonged exercise, mtDNA/nDNA tended to decline (n=10; P=0.083), but was reduced after 3-repeated high intensity exercise bouts (0.900±0.049 vs. 1.067±0.071 at baseline; n=7; P=0.013). Our findings indicate that prolonged and short repeated intense exercise can lead to significant reductions in human skeletal muscle mtDNA content, which might function as a signal stimulating mitochondrial biogenesis with exercise training.