Mitochondria are essential for life, and maintaining mitochondrial function is important to prevent the progression of many age-related diseases. We have shown that physical activity can increase mitochondrial respiration (1) and many of the proteins associated with mitochondrial biogenesis (2, 3). However, little is known about the effects of intensified training on mitochondrial function (ie mitochondrial respiration) and proteins associated with mitochondrial biogenesis. Furthermore, the effects of detraining on these exercise-induced mitochondrial adaptations are unknown. Ten active men (Peak VO2=47.0 mL/kg/min) took part in an interval training program (IT) 3x/wk for 4 wk, an intensified cycling training program (INT), in which they performed interval training 2x/day for 20 consecutive days, and a 2-wk detraining period (DT) consisting of 6 training sessions. Resting muscle biopsies were taken from the vastus lateralis muscle before the study and after the IT, INT and DT phases. Sub-maximal (0.25 mM) and maximal (5 mM) ADP-stimulated mitochondrial respiration was determined on permeabilised muscle fibers. The relative abundance of the five complexes of the electron transport system (ETS), and proteins associated with mitochondrial biogenesis, was analysed in duplicate via Western blotting. One-way ANOVA, with repeated measures for time, were used to test for the effects of training and detraining on all variables; the level of statistical significance was set at P < 0.05. There were no changes in mitochondrial respiration following IT. However, maximal ADP-stimulated and non-coupled mitochondrial respiration increased significantly, by 47% and 48% respectively, during the INT phase, and decreased significantly during the DT phase (17% and 22% respectively); sub-maximal ADP-stimulated mitochondrial respiration showed no significant change during any phase. Following IT, the protein content of the five complexes of the ETS did not change significantly, although there was a trend for all to increase. The protein content of all five complexes was significantly elevated from baseline at the end of INT, with Complex II increasing by ~40%, Complex I and IV increasing by ~20% and Complex V and III increasing by ~10%. Following DT all complexes returned to baseline, except for complex V. There were significant increases in most proteins associated with mitochondrial biogenesis following INT (eg PGC-1α, p53, NRF1, NRF2, TFAM), with some then decreasing towards baseline values following DT (eg p53 and TFAM). Our study demonstrates that three weeks of INT is sufficient to significantly increase mitochondrial respiration, as well as the relative abundance of ETS proteins, and proteins associated with mitochondrial biogenesis. A large proportion of these adaptations are reversed following 2 weeks of detraining, highlighting the remarkable plasticity of skeletal muscle mitochondria.