Introduction Hypovolemia and hypovolemic shock are life-threatening conditions that occur in numerous clinical scenarios. Near-infrared spectroscopy (NIRS) has been widely explored, successfully and unsuccessfully, in an attempt to use it as an early detector of hypovolemia by measuring tissue oxygen saturation (StO2). In order to investigate the measurement site dependence and probe dependence of NIRS in response to hemodynamic changes, such as hypovolemia, we applied a simple cardiovascular challenge: a posture change from supine to upright, causing a decrease in stroke volume (as in hypovolemia) and a heart rate increase in combination with peripheral vasoconstriction to maintain adequate blood pressure. Methods Multi-depth NIRS was used in nine healthy volunteers to assess changes in StO2 in the thenar and forearm in response to the hemodynamic changes associated with a posture change from supine to upright. Results A posture change from supine to upright resulted in a significant increase (P < 0.001) in heart rate. Thenar StO2 did not respond to the hemodynamic changes following the posture change, whereas forearm StO2 did. Forearm StO2 was significantly lower (P < 0.001) in the upright position compared to supine for all probing depths. Conclusions The primary findings in this study were that forearm StO2 is a more sensitive parameter to hemodynamic changes than thenar StO2 and that the depth at which StO2 is measured is of minor influence. Our data support the use of forearm StO2 as a sensitive parameter for the detection of central hypovolemia and hypovolemic shock in (trauma) patients.