To evaluate whether there is a refractory period (RP) after hyperventilation challenge, we compared the bronchoconstrictive response to repetitive eucapnic voluntary hyperventilation challenge with dry, room temperature air (EVH). The serial challenges were identical with 11 known asthmatics hyperventilating for 6 min at a target minute ventilation of 30 times their FEV1 measured before either challenge. There was a significant difference between the mean postchallenge fall in FEV1 to the initial EVH challenge (27.4 +/- 9.8%) and the response to the second EVH challenge (16.1 +/- 5.9%) (p = 0.0001), indicating refractoriness. We then evaluated whether 6 min of uninterrupted EVH challenge gives a similar bronchospastic response to that which results from an equivalent (by total minute ventilation) interrupted challenge of 2 min of EVH repeated three times. The mean post-challenge fall in FEV1 in response to this interrupted challenge was 18.9 +/- 10.6%, which was significantly different from the 27.4% fall in response to 6 consecutive minutes of EVH (p = 0.036). This study demonstrates that there is a refractory period after repeated EVH challenges and this refractoriness affects the response to interrupted, or dosed, EVH challenge.