Two way valve mouthpiece

All participants arrived at the laboratory following an overnight fast and had refrained from alcohol and exercise for ≥ 24 h, and caffeine for ≥ 12 h, prior to the visit. Following a minimum of 20 min supine rest, measurements of middle cerebral artery velocity (MCAv) were obtained using transcranial Doppler ultrasound (TCD) following standardised procedures (Willie et al. 2011 (link)). Two 2-MHz Doppler probes (Spencer Technologies, Seattle, USA) were placed over the temporal window and adjusted until an optimal signal was identified and held in place using a Marc 600 head frame (Spencer Technologies, Seattle, USA). Beat-to-beat blood pressure was recorded using a Finometer (Model 1, Finapres Medical Systems BV, Amsterdam, The Netherlands). Participants were fitted with a photoplethysmographic cuff on the right index finger, and the output was corrected by referencing the cuff to heart level using a height correction unit and heart rate (HR) acquired from a 3-lead electrocardiogram. Partial pressure of end tidal carbon dioxide (P_ETCO₂) was continuously monitored by instrumenting participants with a two-way valve mouthpiece (Hans Rudolph) connected to a calibrated gas analyser (ML206 ADinstruments, Colorado Springs, USA). All data were sampled at 50 Hz with the data acquisition system PowerLab via the interface LabChart 7 (ADinstruments, Colorado Springs, USA).

Following 20 min rest in the supine position, bilateral MCAv’s were continuously measured through the temporal window using transcranial Doppler ultrasonography (TCD). Two 2-MHz Doppler probes (Spencer Technologies, Seattle, USA) were adjusted until an optimal signal was identified and held in place using a Marc 600 head frame (Spencer Technologies, Seattle, USA). Once the optimal MCAv signal was attained, the probe location and machine settings (depth, gain and power) were recorded to identify the same imaging site for the second testing session. Participants were instrumented with a two-way valve mouthpiece (Hans Rudolph) from which end tidal CO₂ (P_ETCO₂) was measured using a calibrated gas analyser (ML206 ADinstruments, Colorado Springs, USA). Continuous beat-by-beat blood pressure was obtained from a digit (Finapres, Amsterdam, Netherlands) and heart rate acquired from a 3 lead electrocardiogram. All data was sampled at 50 Hz with the data acquisition system PowerLab via the interface LabChart 7 (ADinstruments, Colorado Springs, USA).