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 O<sub>2</sub> reactivity as percentage change of cerebral blood flow velocity (CBFV), as absolute change in the concentrations (measured in µmol/l) of oxygenated (HbO<sub>2</sub>), deoxygenated (Hb) and total hemoglobin (HbT), and as change in the tissue oxygenation index (TOI) during inhalation of 100% oxygen. CO<sub>2</sub> reactivity was calculated as percentage change of CBFV (NCR), as absolute change in the concentrations of HbO<sub>2</sub>, Hb, and HbT (µmol/l), and as change in TOI (%) per 1% increase in end-tidal CO<sub>2</sub>. 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 HbO<sub>2</sub> (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). CO<sub>2</sub> reactivity was: NCR left 25.4 ± 14.7%; NCR right 25.9 ± 13.4%; HbO<sub>2</sub> 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%. O<sub>2</sub> reactivity in TCD did not correlate with NIRS reactivities (Pearson p > 0.05), but NCR did correlate with changes in HbO<sub>2</sub>, 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.