For adequate development and functioning of the neonatal brain, sufficient oxygen (O2) should be available. With a fast sampling (fs > 50 Hz) continuous wave NIRS device, arterial (SaO2) and venous (SvO2) saturation can be measured using the physiological fluctuations in the oxyhemoglobin (O2Hb) and total hemoglobin (tHb) concentrations due to heart action and respiration. Before using this technique in a neonatal setting, the method was verified on adult volunteers (n=7) by decreasing inspired oxygen down to an arterial saturation of 70% using a pulse oximeter as reference. NIRS optodes were placed on the left forehead; the pulse oximeter sensor was placed on the right forehead. The experiments were repeated with different optode spacings. SaO2 and SvO2 were determined using the ratio between the O2Hb and tHb value in the amplitude spectrum at the heart rate and respiration rate, respectively. A good agreement between calculated SaO2 and reference SaO2 from pulse oximetry was found (bias range-3.5% to 5.2%, SD of the residuals 1.3% to 3.5%). Optode spacing of 15 mm yielded a negative bias compared to optode spacing of 45 mm. It was not always possible to calculate SvO2 because the respiration peak could not always be detected. © Springer Science+Business Media, LLC 2009.