Velocity-selective arterial spin labelling (VSASL) MRI is insensitive to prolonged arterial transit time. This is an advantage over other arterial spin labelling schemes, where long arterial transit times can lead to bias. Therefore, VSASL can be used with greater confidence to study perfusion in the presence of long arterial transit times, such as in the ageing brain, in vascular pathologies, and cancer, or where arterial transit time changes, such as during measurement of cerebrovascular reactivity (CVR). However, when calculating perfusion (cerebral blood flow, CBF, in the brain) from VSASL signal, it is assumed that a vascular crushing module, defining the duration of the bolus, is applied before the arrival of the trailing edge. The early arrival of the trailing edge of the labelled bolus of blood will cause an underestimation of perfusion. Here, we measure bolus duration in adult, healthy human brains, both at rest and during elevated CBF during CO 2 breathing (5% inspired CO 2). Grey matter bolus duration was of 2.20 ± 0.35 s/2.22 ± 0.53 s/2.05 ± 0.34 s (2/3/4 cm/s v cutoff) at rest, in close agreement with a prior investigation. However, we observed a significant decrease in bolus duration during hypercapnia, and a matched reduction in CVR above a labelling delay of approximately 1.2 s. The reduction in CVR and bolus duration was spatially heterogenous, with shorter hypercapnic bolus durations observed in the frontal lobe (1.31 ± 0.54 s) and temporal lobes (1.36 ± 0.24 s), compared to the occipital lobe (1.50 ± 0.26 s). We place these results in the context of recommendations from a recent consensus paper, which recommends imaging 1.4 s after the label, which could lead to CBF underestimation in conditions with fast flow or during CVR measurements. These results can be used to inform the experimental design of future VSASL studies, to avoid underestimating perfusion by imaging after the arrival of the trailing edge of the labelled bolus.