Sex differences in response to hypovolemia are still an open issue, which is readdressed here by exploiting the potential of near-infrared spectroscopy (NIRS) to monitor the response of lower body negative pressure (LBNP) in upper and lower limbs. In 28 subjects in a supine position, non-invasive arterial blood pressure was continuously monitored along with NIRS parameters from the forearm and thigh during randomized 90 s exposure to LBNP at −10, −20, −30, and −40 mmHg, followed by one 5 min exposure to −30 mmHg. LBNP did not affect arterial blood pressure, slightly increased the heart rate, and affected blood volume in both limbs (p < 0.005). Slopes of tissue oxygenation and deoxy-hemoglobin revealed pressure (p < 0.001) and sex (p < 0.05) dependences for the vasoconstrictive response to LBNP in both arms and legs, with some evidence of larger vasoconstriction in legs. Most variables reached a stable value within 90 s in the arm, while longer time courses were observed in the leg. NIRS is a valuable methodology to detect early LBNP-induced hemodynamic changes, providing that blood volume and blood flow contribution are discriminated. A comparative analysis of time courses proved useful in revealing stronger vasoconstrictive responses in males than in females and in lower limbs than in upper limbs. The same approach could be applied to other experimental contexts.