High-intensity interval training (HIT) performed at an "all-out" intensity is a potent stimulus for mitochondrial biogenesis in human skeletal muscle (Gibala et al. J Physiol. 575: 901-911, 2006). Six sessions of HIT over 2 wk (4-6 maximal 30 s cycle sprints with 4 min of recovery per session) increased mitochondrial enzyme activity to the same extent as traditional endurance training (90-120 min of moderate-intensity cycling per session). These HIT-induced adaptations appear linked to repeated transient increases in the transcriptional coactivator PGC-1α (Gibala et al. J Appl Physiol. 106: 929-934, 2009). Thus, a few minutes of very high intensity exercise performed in an intermittent fashion, and interspersed with brief rest/recovery periods is sufficient to increases PGC-1α(acutely) and muscle oxidative capacity (chronically) in human skeletal muscle. However the relative importance of the "pulsed" nature of the HIT stimulus remains unclear. PURPOSE: To determine whether a single bout of all out exercise would stimulate an increase in PGC-1αmRNA expression similar to an intermittent maximal cycling protocol matched for total work output. METHODS: Eight men (22±1 y, 48±2 mL·kg−1·min−1) performed two exercise trials in random order ≥1 wk apart. During one session, subjects performed 4 × 30 s all out sprints separated by 4 min of rest (HIT). In the other trial, subjects performed an isovolumetric amount of work (67.0±2.4 kJ; IsoV) as the HIT trial, but in a single continuous all-out effort. RESULTS: Mean power averaged over the 4 sprints was 557±16 W, whereas mean power during the single bout was 287±18 W. Thus, mean time to completion in IsoV was ∼4 min (3:58±0:09 min:s; range: 3:23-4:30 min:s), near double that of the 4 × 30 s HIT trial (excluding recovery, p<0.05). However, when HIT recovery was included, total time commitment of the IsoV trial was only ∼30% of HIT. RT-PCR analysis of skeletal muscle biopsies revealed a similar ∼3.1-fold increase in PGC-1αmRNA 3 h post-exercise compared to pre-exercise, with no differences between trials (p<0.05, main effect for time). CONCLUSIONS: These findings indicate that augmentations in PGC-1αmRNA expression are achieved by very low volumes of all-out exercise whether performed continuously or intermittently. It appears that as little as 3-5 min of all out continuous exercise may be sufficient to stimulate mitochondrial biogenesis in human skeletal muscle. Supported by NSERC.