## 1 Introduction  
Lower body negative pressure (LBNP) has been adopted since the 1960s as a means to induce hypovolemic stimuli in the absence of the hydrostatic gradients that develop with maneuvers, such as standing or head-up tilt. For this reason, it has been conveniently employed for the investigation of autonomic reflexes, namely, the cardio-pulmonary and the arterial baroreceptor reflexes, and their capacity to prevent cardiovascular collapse [1]. In general terms, it is agreed that women present a lower tolerance to hypovolemic stimuli then men [2, 3, 4] although the underlying mechanisms are still debated [5]. For instance, contrasting results concern the extent of peripheral vasoconstriction, which has been reported to be both stronger [2] and lower in women than men [6, 7]. In this respect, near-infrared spectroscopy (NIRS) represents an attractive non-invasive methodology for the investigation of peripheral vascular effects, allowing the simultaneous detection of changes in blood volume and tissue oxygenation as well as the inference of changes in blood flow and vascular resistance in different body areas. In fact, tissue oxygenation essentially reflects the balance between blood flow and metabolism, providing that the blood volume remains constant [8]; thus, changes in blood flow can be inferred from corresponding changes in tissue oxygenation, as reported in different conditions [9; 10; 11]. However, the interpretation of NIRS signals is complicated by several factors, including (1) dependence on contributions from superficial (cutaneous) and deep (muscle/brain) tissue layers, which may respond differently to autonomic and environmental challenges [12; 13], and (2) dependence on changes in blood volume, perfusion, and metabolism, which may occur at the same time and complicate the interpretation of NIRS signals [14]. This latter issue is particularly relevant in LBNP studies in which marked changes in blood volume take place (increases and decreases in lower and upper body areas, respectively) along with vascular reflexes affecting local perfusion. Moreover, it must be reiterated that although this blood volume shift is likely to mainly affect the largest and most compliant veins, these may also affect NIRS measurements, as recently demonstrated during venous dilatation produced by venous occlusion tests [15] and remote external heating [16]. On this basis, it is crucial to know the full time course of NIRS signals for their correct interpretation. However, to the best of our knowledge, with the exception of a few original recordings from single subjects, the time course of blood volume and oxygenation changes in response to LBNP has never been reported. In addition, most studies have adopted LBNP protocols in which the pressure progressively decreased, usually in steps of 10 mmHg and 5 min duration. Therefore, the hemodynamic effects observed at a given negative pressure are not representative of that pressure level, being affected by LBNP exposure to all previous pressure steps. To precisely relate the hemodynamic changes to the different pressure levels, distinct LBNP stimuli should be separated by resting intervals (with no LBNP applied). We hypothesized that (1) by examining the time course of NIRS signals in response to distinct LBNP stimuli of different magnitudes, it would be possible to identify the time intervals in which tissue blood volume exhibits the fastest transients and those in which it is relatively stable, thus allowing the discrimination of blood volume displacements from changes in blood flow; (2) upper and lower limbs would exhibit opposite changes in blood volume but a similar reduction in tissue oxygenation as a result of vasoconstriction in a pressure-dependent way; and (3) females would exhibit weaker signs of vasoconstriction than males, which would explain their lower tolerance to the hypovoicmic challenge.