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Hypercapnia challenge (e.g. inhalation of CO2) has been used in calibrated fMRI as well as in the mapping of vascular reactivity in cerebrovascular diseases. An important assumption underlying these measurements is that CO2 is a pure vascular challenge but does not alter neural activity. However, recent reports have suggested that CO2 inhalation may suppress neural activity and brain metabolic rate. Therefore, the goal of this study is to propose and test a gas challenge that is truly “iso-metabolic,” by adding a hypoxic component to the hypercapnic challenge, since hypoxia has been shown to enhance cerebral metabolic rate of oxygen (CMRO2). Measurement of global CMRO2 under various gas challenge conditions revealed that, while hypercapnia (P = 0.002) and hypoxia (P = 0.002) individually altered CMRO2 (by −7.6 ± 1.7% and 16.7 ± 4.1%, respectively), inhalation of hypercapnic-hypoxia gas (5% CO2/13% O2) did not change brain metabolism (CMRO2 change: 1.5 ± 3.9%, P = 0.92). Moreover, cerebral blood flow response to the hypercapnic-hypoxia challenge (in terms of % change per mmHg CO2 change) was even greater than that to hypercapnia alone (P = 0.007). Findings in this study suggest that hypercapnic-hypoxia gas challenge may be a useful maneuver in physiological MRI as it preserves vasodilatory response yet does not alter brain metabolism. [ABSTRACT FROM AUTHOR]