Background: Spatially resolved spectroscopy is a recently developed technique for noninvasive monitoring of cerebral tissue oxygenation using the photon diffusion theory. Methods: We studied this technique with a new, commercial near-infrared spectroscopy (NIRS) device during vasomotor reactivity tests in 28 healthy volunteers (mean age 31.0 years; SD 10.6 years) and compared it with values assessed by the modified Beer-Lambert law and indices from simultaneous transcranial Doppler sonography of both middle cerebral arteries. We measured O2 reactivity as percentage change of cerebral blood flow velocity (CBFV), as absolute change in the concentrations (measured in µmol/l) of oxygenated (HbO2), deoxygenated (Hb) and total hemoglobin (HbT), and as change in the tissue oxygenation index (TOI) during inhalation of 100% oxygen. CO2 reactivity was calculated as percentage change of CBFV (NCR), as absolute change in the concentrations of HbO2, Hb, and HbT (µmol/l), and as change in TOI (%) per 1% increase in end-tidal CO2. Results: One hundred percent oxygen inhalation lead to a decrease in CBFV (mean ± SD: left –8.0 ± 7.0%, p = 0.000; right –9.6 ± 7.6%, p = 0.000), an increase in HbO2 (0.99 ± 1.07 µmol/l), Hbdiff (2.23 ± 1.72 µmol/l), and TOI (3.1 ± 1.5%), and a decrease in Hb (–1.22 ± 0.74 µmol/l), significant from baseline values (p = 0.0000). CO2 reactivity was: NCR left 25.4 ± 14.7%; NCR right 25.9 ± 13.4%; HbO2 1.99 ± 0.97 µmol/l; Hb –1.24 ± 0.81 µmol/l; HbT 0.81 ± 1.0 µmol/l, and TOI 3.7 ± 2.2%. O2 reactivity in TCD did not correlate with NIRS reactivities (Pearson p > 0.05), but NCR did correlate with changes in HbO2, Hb, and TOI (Pearson p < 0.01). TOI was closely related to indices derived from the Beer-Lambert law (Pearson p < 0.03), but not with mean arterial blood pressure or skin blood flow during vasomotor reactivity tests. Conclusion: Spatially resolved spectroscopy provides an encouraging, noninvasive new tool to study cerebral tissue oxygenation during vasomotor reactivity tests consistent with physiological changes.