We tested the hypothesis that priming exercise would speed V˙O2 kinetics during treadmill running. Eight subjects completed a square-wave protocol, involving two bouts of treadmill running at 70% of the difference between the running speeds at lactate threshold (LT) and V˙O2 max, separated by 6-min of walking at 4 km h−1, on two occasions. Oxygen uptake was measured breath-by-breath and subsequently modelled using non-linear regression techniques. Heart rate and blood lactate concentration were significantly elevated prior to the second exercise bout compared to the first. However, V˙O2 kinetics was not significantly different between the first and second exercise bouts (mean ± S.D., phase II time constant, Bout 1: 16 ± 3 s vs. Bout 2: 16 ± 4 s; V˙O2 slow component amplitude, Bout 1: 0.24 ± 0.10 L min−1 vs. Bout 2: 0.20 ± 0.12 L min−1; mean response time, Bout 1: 34 ± 4 s vs. Bout 2: 34 ± 6 s; P > 0.05 for all comparisons). These results indicate that, contrary to previous findings with other exercise modalities, priming exercise does not alter V˙O2 kinetics during high-intensity treadmill running, at least in physically active young subjects. We speculate that the relatively fast V˙O2 kinetics and the relatively small V˙O2 slow component in the control (‘un-primed’) condition negated any enhancement of V˙O2 kinetics by priming exercise in this exercise modality.