Background and Purpose-The relationship between middle cerebral artery (MCA) flow velocity (CFV) and cerebral blood now (CBF) is uncertain because of unknown vessel diameter response to physiological stimuli. The purpose of this study was to directly examine the effect of a simulated orthostatic stress (lower body negative pressure [LBNP]) as well as increased or decreased end-tidal carbon dioxide partial pressure (PETCO2) on MCA diameter and CFV. Methods-Twelve subjects participated in a CO2 manipulation protocol and/or an LBNP protocol. In the CO2 manipulation protocol, subjects breathed room air (normocapnia) or 6% inspired CO, (hypercapnia), or they hyperventilated to approximate to 25 mmHg PETCO2 (hypocapnia). In the LBNP protocol, subjects experienced 10 minutes each of -20 and -40 mm Hg lower body suction. CFV and diameter of the MCA were measured by transcranial Doppler and MRI, respectively, during the experimental protocols. Results-Compared with normocapnia, hypercapnia produced increases in both PETCO2 (from 36+/-3 to 40+/-4 mm Hg, P<0.05) and CFV (from 63+/-4 to 80+/-6 cm/s, P<0.001) but did not change MCA diameters (from 2.9+/-0.3 to 2.8+/-0.3 mm). Hypocapnia produced decreases in both PETCO2 (24+/-2 mmHg, P<0.005) and CFV (43+/-7 cm/s, P<0.001) compared with normocapnia, with no change in MCA diameters (from 2.9+/-0.3 to 2.9+/-0.4 mm). During -40 mm Hg LBNP, PETCO2 was not changed, but CFV (55+/-4 cm/s) was reduced from baseline (58+/-4 cm/s, P<0.05), with no change in MCA diameter. Conclusions-Under the conditions of this study, changes in MCA diameter were not detected. Therefore, we conclude that relative changes in CFV were representative of changes in CBF during the physiological stimuli of moderate LBNP or changes in PETCO2.