The responsiveness of the cerebrovasculature to alterations in arterial content of carbon dioxide (PCO2) is a common functional test to assess brain health (i.e., CBF-CO2 responsiveness). The most common and simplest test to assess CBF-CO2 responsiveness is the inhalation of one concentration of CO2 via a Douglas bag open circuit (e.g., 5% CO2 in air). Across the studies using this approach a range of durations have been used to induce this stimulus-response, from 1 to 5 min. However, given the known kinetics of this response, a 1 min response test may represent something different to that of a 5 min test. Further, given the interaction between the ventilatory and vascular responses with the open-circuit technique, the ventilatory response may also affect the consistency between the different stimulus durations for the CBF-CO2 responsiveness measure. The purpose of this study was to examine whether 1, 2, 4 and 5 mins of CO2 stimulus gave the same CBF-CO2 responsiveness measure. Fourteen healthy volunteers were recruited. Following a full familiarisation visit, participants attended the laboratory for one experimental trial. Participants lay supine for ~20 min while being instrumented, following which they completed a 5 min resting baseline. In a randomised order, participants then completed four durations (1, 2, 4 and 5 minutes) of 5% CO2 (in air) inhalation via the open-circuit Douglas bag method, with 5 min recovery between. Bilateral middle cerebral artery velocity (MCAv, transcranial Doppler), mean arterial pressure (MAP, Finometer), ventilation (VE) and partial pressure of end-tidal carbon dioxide (PETCO2) were recorded continuously. CBF-CO2 responsiveness and ventilatory sensitivity to CO2 (VE-CO2) were calculated as change from baseline (relative and absolute, respectively) per mm Hg change in PETCO2. We compared values calculated from the final 30 s of each CO2-stimulus duration. A repeated-measures ANOVA (Bonferroni corrected) was used to test differences between durations. We observed no statistical difference (p=0.15) across the different durations of CO2-stimulus for CBF-CO2 responsiveness (bilateral Doppler data pooled (mean±SD): 2.4±1.2, 2.4±1.1, 2.3±1.1, 2.3±1.1 %ΔMCAv/mm Hg PETCO2, for 1, 2, 4 and 5 min respectively), despite observable differences in the MCAv-response profile between durations. For example, a steady-state profile typified the 4 and 5 min tests, whereas the 1 min test tended to peak in the final seconds of the stimulus. Further, while grouped CBF-CO2 responsiveness data appear consistent across durations, there was variation within individuals across the four stimulus durations; CoV ranging from 8 – 45% between individuals. In contrast, VE-CO2 sensitivity increased ~3-fold from the 1 min to the 4- and 5-min test durations (0.3±0.2, 0.6±0.3, 0.9±0.3, 1.0±0.4 L·min-1/mm Hg, for 1, 2, 4 and 5 min respectively; p<0.01).