Alterations to cerebral blood flow (CBF) have been implicated in diverse neurological conditions. Near-infrared spectroscopy (NIRS)-measured regional cerebral tissue oxygen saturation (rSO2) provides an estimate of oxygenation of interrogated cerebral volume useful in identifying variations in oxygen supply to cerebral tissue and in monitoring cerebrovascular function. CO2-inhalation-based hypercapnic breathing challenges were used to simulate CBF dysregulation, utilizing NIRS to monitor the CBF autoregulatory response. A breathing circuit was designed to administer CO2-compressed air mixtures and assess CBF regulatory responses to hypercapnia in 26 healthy young adults. One to three hypercapnic challenges of 5 or 10 min duration were delivered to each subject while continuously monitoring rSO2, partial pressure of end tidal CO2 (PETCO2), and vital signs. Change in rSO2 (ΔrSO2) during CO2 inhalation positively correlated to ΔPETCO2 (R2=0.40). Grouping subjects into three exercise factor levels (h/week), (1) 0, (2) <0 and <10, and (3) <10 showed significantly greater ΔrSO2 responses to CO2 challenges for level 3 subjects but similar PETCO2 responses for the three groups. Exercising greater than 10 h/week may produce a higher resting cerebrovascular reactivity (CVR) to CO2 inhalation. Establishing baseline values of rSO2 and CVR to CO2 may aid in early detection of CBF changes.