Background and Purpose-The relationship between middle cerebral artery (MCA) flow velocity (CFV) and cerebral blood flow (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 (P ET CO 2 ) on MCA diameter and CFV. Methods-Twelve subjects participated in a CO 2 manipulation protocol and/or an LBNP protocol. In the CO 2 manipulation protocol, subjects breathed room air (normocapnia) or 6% inspired CO 2 (hypercapnia), or they hyperventilated to Ϸ25 mm Hg P ET CO 2 (hypocapnia). In the LBNP protocol, subjects experienced 10 minutes each of Ϫ20 and Ϫ40 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 P ET CO 2 (from 36Ϯ3 to 40Ϯ4 mm Hg, PϽ0.05) and CFV (from 63Ϯ4 to 80Ϯ6 cm/s, PϽ0.001) but did not change MCA diameters (from 2.9Ϯ0.3 to 2.8Ϯ0.3 mm). Hypocapnia produced decreases in both P ET CO 2 (24Ϯ2 mm Hg, PϽ0.005) and CFV (43Ϯ7 cm/s, PϽ0.001) compared with normocapnia, with no change in MCA diameters (from 2.9Ϯ0.3 to 2.9Ϯ0.4 mm). During Ϫ40 mm Hg LBNP, P ET CO 2 was not changed, but CFV (55Ϯ4 cm/s) was reduced from baseline (58Ϯ4 cm/s, PϽ0.05), 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 P ET CO 2 .