The results described hemodynamic responses of temporalis and masseter muscles to different stressors, which effectively increased sympathetic activity, as indicated by the significant increase in ABP in some cases accompanied by an increase in heart rate and cardiac output. In particular, tissue oxygenation significantly increased in both muscles during mental arithmetic test and isometric handgrip, accompanied by increased or unchanged blood volume which indicates increased muscle perfusion during these tests (Boushel and Piantadosi 2000; Fadel et al. 2004; Rashid and Roatta 2022). No significant changes were instead observed during cold pressor test, apnea, and post-handgrip muscle ischemia. However, none of the tested conditions provided evidence of sympathetic-induced vasoconstriction. Both temporalis and masseter muscles consistently exhibited similar response patterns in all tested conditions.

It is well known that sympathetic activation may result in skin vasodilation in the facial area (Vassend and Knardahl 2005). A number of NIRS studies also reported dilatory responses (increased oxygenation) in the masseter muscle in response to certain stress stimuli, e.g., cold pressor test (Maekawa et al. 1998), and mental calculation (Hidaka et al. 2004), although classical methodologies did not discriminate skin from muscle contribution (Grassi and Quaresima 2016). As discussed in more detail below, this can be achieved by techniques such as spatially-resolved spectroscopy which effectively excludes contribution from the skin (Messere and Roatta 2013) and recently allowed us to prove that several types of stress result in specific dilatation of the masseter muscle, in contrast to limb muscles where marked vasoconstriction may take place (Rashid and Roatta 2022). This latter study was the first to directly compare hemodynamic stress responses in head muscles and included an extensive review of the recent literature, although head muscles were limited to the masseter muscle (Rashid and Roatta 2022). Limb muscles were no longer examined in the present study but sympathetic constrictory responses or increases in muscle sympathetic nerve activity in these muscles have consistently been reported in response to several stressors, including cold pressor test (Wray et al. 2007; Jacob et al. 2021; Coovadia et al. 2022), post-exercise muscle ischemia (Saito et al. 2000; Tokizawa et al. 2006), and acute pain stimuli (Burton et al. 2016).

The results of the present study confirm the different pattern of head muscles, now including the temporalis muscle. We are not emphasizing the dilatory responses observed during mental arithmetic test and isometric handgrip, as these are known to take place in limb muscles as well (Rusch et al. 1981; Carter et al. 2005; Rashid and Roatta 2022). Unexpectedly, the significant TOI increase in response to cold pressor test and post-handgrip muscle ischemia previously observed for the masseter muscle (Rashid and Roatta 2022) was no longer observed in the present study. While the absence of response to cold pressor test could be partly attributed to the milder stimulus intensity, in terms of water temperature (10 instead of 8 °C) and duration (1 instead of 2 min), the response to post-handgrip muscle ischemia has no apparent explanation except that the subject position was sitting instead of supine. Incidentally, both active and passive dilatory responses have found to be attenuated above as compared to below heart level (Trinity et al. 2011; Jasperse et al. 2015; Seddone et al. 2020). It is possible that the unfavorable hydrostatic gradient affecting the head region or the increased sympathetic tone have attenuated the hyperemic response in the sitting position, compared to the supine. In fact, in human skeletal muscles, competing vascular actions are exerted by the dilatory action of circulating adrenaline and the constrictory action of the neurally released noradrenaline (Montoya et al. 1997; Terakawa and Ichinohe 2012). In this respect, it is interesting to observe that a poor positive correlation was observed between ΔTOI and ΔABP in mental arithmetic test and isometric handgrip; while a strong negative correlation resulted in post-handgrip muscle ischemia. This observation fits with the concept of prevailing adrenaline secretion during mental arithmetic test and isometric handgrip (Goldberg et al. 1996; Joyner and Dietz 2003; Rashid and Roatta 2022) and prevailing noradrenaline constriction in post-handgrip muscle ischemia: the correlation is poor in the first case; whereby ABP is largely mediated by the increase in cardiac output (significantly increased in these tests). On the contrary during post-handgrip muscle ischemia, ΔABP is mainly mediated by the increased vasoconstriction, being cardiac output unchanged or even decreased (Rashid and Roatta 2022). On this basis, it is plausible to speculate that the baroreflex-mediated increase in sympathetic vasconstrictory tone, associated with the sitting position, has shifted the balance and attenuated the stress-related dilatory responses, compared to the supine position. Dedicated studies re-testing the responses to the same stressor in the same subjects in the two body postures may be designed to test this hypothesis.

Although simultaneous monitoring of a limb muscle was not performed in this study, a relevant outcome is that temporalis and masseter muscle exhibit consistently similar responses to the variety of employed stressors. In fact, this observation supports the hypothesis put forward in our previous study that muscle blood flow is differentially controlled in limb and head muscles. Very few studies investigating differences in blood flow control of lower and upper limb muscles evidenced stronger sympathetic constrictor effects in lower limbs (Eklund and Kaijser 1976; Rusch et al. 1981), we speculate that the constrictory sympathetic action is modulated according to the hydrostatic gradients of the body with erect posture, i.e., the constrictory action is stronger where hydrostatic load and blood pressure are higher. Whether this effect is mediated by differences in sympathetic neural drive and/or differences in the density and distribution of adrenergic receptors subtypes remains to be investigated.

A major limitation of classical NIRS measurement, based on the modified Beer–Lambert methodology is that the contribution from superficial cutaneous layers cannot be discriminated the contribution from deeper layers, e.g., brain or muscle. Spatially-resolved spectroscopy was proven to be particularly effective in focusing the measurement in deep layers as documented in several studies for both cerebral (Canova et al. 2011; Moerman et al. 2022) and muscle monitoring (Messere and Roatta 2013; Grassi and Quaresima 2016). In particular, we showed that contrary to standard NIRS, spatially-resolved spectroscopy was neither affected by local increases in cutaneous microcirculation (Messere and Roatta 2013) nor by increased blood flow in superficial veins (Messere and Roatta 2015), provoked by warming of local or remote distal areas, respectively. For this reason, spatially-resolved spectroscopy was adopted in this study. Besides excluding interference from the skin, NIRS monitoring of the temporal muscle also requires to exclude possible contributions from the deeper cerebral tissue. However, the depth of the sample volume is proportional to the inter-optode distance (about 50% of it). In a previous methodological study, we showed that reducing the inter-optode distance from 4 to 3 cm effectively prevented the influence of cerebral hemodynamic changes, as produced by hyperventilation, on NIRS monitoring of the temporal muscle (Rashid and Roatta 2023). Though, that study also evidenced the placement of the NIRS probe could be critical in some subjects, probably due to the fact that the temporalis muscle becomes too thin and superficial under the hair-free area of the temple, in which case spatially-resolved spectroscopy measurements would not adequately sample the muscle tissue, thus resulting in possible underestimation of the hemodynamic response. We cannot exclude that this could have happened also in the present study. In fact, we observed in a few subjects that changes in tissue oxygenation were considerably weaker than changes in oxygenated and deoxygenated hemoglobin.

An additional limitation of this study is the low sample size, which prevented analysis of sex-related differences. Considered the sex-related prevalence of clinical manifestations such as headache and temporomandibular disorders, the issue is worth to be addressed in future investigations. Finally, we recruited the subjects from a young and healthy student population, we did not assess psychological traits that could affect the reactivity to the different stressor, although this relation this not trivial; e.g., with regard to the pain stress, it has been shown that the autonomic activation is psychogenic in nature, but its variability among individual appeared to be uncorrelated with the anxiety score (Burton et al. 2016).

To conclude, head muscles appear to be similarly controlled by the autonomic nervous system under exposure to different types of stress and generally protected from stress-induced hypoperfusion. From a finalistic point of view, this could be meant to favor activity of jaw muscles, being frequently involved in aggressive/defensive behavior in different animal species. Protection from hypoperfusion may also be beneficial against the development of temporomandibular joint dysfunction (Delcanho 1995; Maekawa et al. 2002; Exposto et al. 2021). It should however be noted that these considerations are based on average responses and that clear vasoconstrictive responses were occasionally observed in some subject, possibly depending on a different balance between beta- and alpha-adrenergic vascular control. In addition, sympathetic reactivity to stress is known to present individual variability related to different factors including, sex, age, the nature and duration of the stressful stimulus (Greaney et al. 2015; Burton et al. 2016; Coovadia et al. 2022). Whether the susceptibility to temporomandibular joint disorders (such as myofascial orofacial pain and temporal headache) development depends on the individual hemodynamic responsiveness to stress needs to be ascertained.