High‐intensity interval training (HIIT) performed in an ‘all‐out’ manner (e.g. repeated Wingate tests) is a time‐efficient strategy to induce skeletal muscle remodelling towards a more oxidative phenotype. A fundamental question that remains unclear, however, is whether the intermittent or ‘pulsed’ nature of the stimulus is critical to the adaptive response. In study 1, we examined whether the activation of signalling cascades linked to mitochondrial biogenesis was dependent on the manner in which an acute high‐intensity exercise stimulus was applied. Subjects performed either four 30 s Wingate tests interspersed with 4 min of rest (INT) or a bout of continuous exercise (CONT) that was matched for total work (67 ± 7 kJ) and which required ∼4 min to complete as fast as possible. Both protocols elicited similar increases in markers of adenosine monophosphate‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase activation, as well as Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) mRNA expression (main effects for time, P ≤ 0.05). In study 2, we determined whether 6 weeks of the CONT protocol (3 days per week) would increase skeletal muscle mitochondrial content to a similar extent to what we have previously reported after 6 weeks of INT. Despite similar acute signalling responses to the CONT and INT protocols, training with CONT did not increase the maximal activity or protein content of a range of mitochondrial markers. However, peak oxygen uptake was higher after CONT training (from 45.7 ± 5.4 to 48.3 ± 6.5 ml kg⁻¹ min⁻¹; P < 0.05) and 250 kJ time trial performance was improved (from 26:32 ± 4:48 to 23:55 ± 4:16 min:s; P < 0.001) in our recreationally active participants. We conclude that the intermittent nature of the stimulus is important for maximizing skeletal muscle adaptations to low‐volume, all‐out HIIT. Despite the lack of skeletal muscle mitochondrial adaptations, our data show that a training programme based on a brief bout of high‐intensity exercise, which lasted <10 min per session including warm‐up, and performed three times per week for 6 weeks, improved peak oxygen uptake in young healthy subjects.