New Findings: What is the central question of this study? We characterized and compared the cardiorespiratory and cerebrovascular responses to the ‘Duffin’ modified hyperoxic CO2 rebreathing test by randomly altering the prior hyperventilation duration. What is the main finding and its importance? Our main finding was that prior hyperventilation duration (1, 3 or 5 min) had no effect on cardiorespiratory and cerebrovascular responses to the hyperoxic rebreathing test, within individuals. These findings suggest that the standard 5 min prior hyperventilation duration used to clear body CO2 stores is unnecessary and can reasonably be shortened to 1 min, reducing protocol times and improving participant comfort. The ‘Duffin’ modified hyperoxic rebreathing test allows investigators to characterize and quantify the ventilatory and cerebrovascular responses to CO2 across a large physiological range, allowing quantification of basal ventilation and the ventilatory recruitment threshold (VRT). Although the standard protocol includes 5 min of prior hyperventilation to clear body CO2 stores, there is no experimental evidence that a full 5 min is required. We hypothesized that there would be no within-individual differences in the cardiorespiratory or cerebrovascular responses to rebreathing with shortened hyperventilation duration prior to hyperoxic rebreathing. Using a rebreathing apparatus, transcranial Doppler ultrasound and beat-to-beat blood pressure monitoring, we tested 19 participants in the supine position using three randomly assigned hyperoxic rebreathing tests with 1, 3 or 5 min of prior hyperventilation. We measured VRT (in Torr CO2), time to VRT (in seconds), central respiratory chemoreflex (breathing frequency, tidal volume and minute ventilation), cerebrovascular (middle and posterior cerebral artery velocity) and cardiovascular (heart rate and mean arterial pressure) responses to CO2 during hyperoxic rebreathing. Using linear regression and repeated-measures ANOVAs, we found no differences in any of the cardiorespiratory or cerebrovascular response magnitudes between trials (P > 0.05). The only difference observed was in the time to VRT (in seconds), whereby 1 min prior hyperventilation duration was shorter (135.4 ± 19.7 s) than with 3 or 5 min prior hyperventilation (176.3 ± 15.1 and 187.2 ± 11.6 s, respectively; P < 0.001). Our findings indicate that 5 min of prior hyperventilation is unnecessary during modified rebreathing when using it to quantify respiratory or cerebrovascular responses and can be reasonably shortened to 1 min, reducing protocol times and improving participant comfort.