## 4 Discussion
### Analysis of Time Courses Reveals Blood Volume and Oxygen Changes
It has been proposed that the myogenic constriction in response to the increase in transmural pressure is primarily responsible for the increase in vascular resistance of lower limbs [29]. However, this mechanism does not explain the similar response to LBNP of upper limbs, not concerned by changes in transmural pressure. The vasoconstriction in upper and, at least in part, in lower limbs must be attributed to increases in sympathetic activity as documented also by direct recording of muscle sympathetic nerve activity in the same conditions [24; 30]. Based on the lack of a visible drops in arterial blood pressure and increases in heart rate, at these low levels of LBNP stimuli (as observed also in the present study), it is generally considered that the sympathetic nervous system is primarily activated by the cardiopulmonary reflex [24; 31], with only minor contributions from the arterial baroreflex [1]. Notably, hemodynamic changes in the upper limbs almost fully develop in about 90 s while accommodation of blood volume in the lower limbs takes more than 5 min. One aspect that remains to be discussed is the transient TOI increase that takes place at the beginning of the LBNP stimulus. In the arm, it is small and short lasting (<20 s) and is likely due to the emptying of the venous compartment, i.e., it does not mean a real increase in oxygenation in the tissues but simply reflects the reduced amount of deoxygenated hemoglobin in the sample volume. The same explanation does not apply to the leg, where venous blood volume increases. The larger and longer-lasting TOI increase in the leg reflects, in fact, a real increase in oxygenation, caused by a transient hyperemia. This phenomenon was documented by [PERSON] et al. [29] who observed a short-lasting (10-15 s) femoral blood flow increase when exposing one leg, inserted into a limb tank, to negative pressure stimuli. The magnitude of the peak hyperemia increased with the magnitude of the negative pressure, almost doubling resting blood flow, at \(-50\) mmHg. Not surprisingly, the effect on tissue oxygenation largely outlasts the duration of the hyperemia, as has been observed with compressive stimuli and short contractions, eliciting hyperemic responses lasting 10-20 s and a TOI increase of up to 100 s in duration [9,32]. In the present data, this TOI increase is particularly prominent and longer lasting in females (see, Figure 4), possibly due to a weaker constrictory response (see male-female comparison below). Failing to discriminate blood volume from oxygenation changes may limit the understanding of the underlying physiological processes, leading to possible misinterpretations. For instance, HHb in the forearm was reported to be unaffected by LBNP up to \(-50\) mmHg [33]. However, the present results clearly indicate a prominent vasoconstriction-induced HHb increase, compensating the initial drop caused by venous emptying. Curiously, since both phenomena are dependent on the magnitude of the LBNP, such compensation is reproduced at the different pressures so that HHb returns close to control levels within 90 s from the beginning of the LBNP, in all conditions (Figure 2). A specular pattern can be exhibited by O2 Hb in lower limbs whereby an initial increase due to blood volume increase and hyperemia is followed by a decrease due to reduced blood flow and increased Hb desaturation. The two changes may compensate each other, leading to an overall non-significant change in O2 Hb (with LBNP up to \(-30\) mmHg) and to the conclusion that vasoconstriction in lower legs does not take place or is delayed compared to other compartments [34]. On the contrary, the present study documents consistent constriction at the thigh already at \(-10\) mmHg, as revealed by TOI and HHb slopes.