All procedures were approved by the York University Research Ethics Board and all participants gave written informed consent. We adhered to the Declaration of Helsinki and Title 45, US Code of Federal Results, Part 46, Protection of Human Subjects, and all subsequent revisions and amendments. Two unique cohorts of participants were recruited as the EndoPAT protocols were completed as a follow-up study to the FMD protocol. The FMD protocol consisted of 10 women and nine men, while the EndoPAT protocol included nine women and eight men (Table 1). Participants were excluded if they suffered from any previously diagnosed cardiovascular or pulmonary diseases. Women must have never taken oral contraceptives or have stopped taking them for a period of at least 3 months prior to participating in the study. Women were also excluded if they had been using a hormonal intrauterine device, contraceptive patches, or any other form of hormonal contraceptives. Women were tested in the early follicular phase during days 2–5 of the menstrual cycle. For 12 h before testing, all participants were asked to refrain from: smoking (e.g., cigarettes, vaping, and marijuana), drinking alcohol, drinking caffeine (e.g., coffee and tea), heavy exercise (including sports, resistance training, and moderate to intense aerobic exercises), and eating fatty foods. Height and weight were measured with a standard stadiometer and age, sex, and weekly episodes of moderate to vigorous exercise were by self-report. Estimated VO₂max was calculated using anthropometrics and the frequency of exercise using the Ainsworth equation (Ainsworth et al., 1993).

Each FMD or EndoPAT protocol (one while supine and one while 70° upright) consisted of baseline measurements, forearm occlusion/ischemia, and reperfusion measurements as described above. A standard blood pressure cuff was placed on the forearm of the right arm and for all trials both hands/arms were kept at the level of the heart. For tilted trials, the EndoPAT trial began immediately upon achieving upright posture (~2 min) whereas the FMD trials began within ~5 min due to the necessity of obtaining an adequate ultrasound image in the upright posture. Since the baseline period for the EndoPAT trial is 5 min and the baseline period for the FMD trials is 2 min, we planned for cuff release at approximately the same time between the trials. Supine and upright trials within each methodology were randomized and conducted with a 30-min break between them to minimize any serial effect of the occlusion periods. The protocol is shown in Figure 1.

Anthropometric data were compared with an unpaired t-test between the sexes. One minute hemodynamic averages were taken at baseline (i.e., 1-min prior to cuff inflation while supine or tilted, as appropriate), and 15 s averages were taken at the time of maximal brachial artery dilation (FMD) or the time of maximal finger blood volume (EndoPAT) after cuff-release/reperfusion. In the tilted posture, the baseline values were taken at least 2 min after onset of tilt. Our lab group previously observed that in healthy young men and women blood pressure stabilized after 2 min of 70° upright tilt (Joshi and Edgell, 2019). Changes in hemodynamics were calculated as the change from baseline to the time of maximum dilation. A two-way mixed model ANOVA was used to compare hemodynamic data across the two postures (sex and posture as factors, posture is a repeated measure). Analysis of vascular variables (i.e., FMD, SR, SR_AUC, and Ln-RHI) was also done via a two-way mixed model ANOVA while accounting for sex and posture (repeated measure) as factors. The normality of distribution was assessed via the Spiro-Wilks test of normality. Tukey’s post hoc analysis was used when significance was found. Correlations between the change in FMD between postures and the blood pressure responses were conducted with linear regression. Significance was defined as p < 0.05. All statistical analyses were performed via Sigmaplot 13.2 (San Jose, California, United States) statistical software. Data in tables and text are presented as Mean ± SD. Data in figures are presented as median and the 25 and 75th percentiles.

We hypothesized that FMD would be lower in the tilt position due to reduced shear stress; however, we found no change in shear stress and an improved FMD while upright. We also did not observe any sex differences in FMD in either posture. While FMD can be influenced by other contributing factors, FMD is typically proportional to shear stress as its primary mechanism (Pyke et al., 2004). However, in the current study, we observed an increased FMD in the upright posture with no change in shear stress and a reduction in blood pressure. Hence, our results suggest a mechanism other than shear stress causing the increased vasodilation. Further, while we hypothesized that a reduction of FMD while upright would be protective of blood pressure while upright, these results suggest that enhanced FMD while upright could in fact be a contributing factor to reductions of blood pressure while upright.

Guazzi et al. (2005) previously explored brachial FMD while 60° upright in a healthy group of primarily men (10/12 men). However, shear stress was not calculated nor was continuous arterial diameter and blood pressure (measures were taken every 15 s). Nonetheless, our results in both women and men correspond with their findings. Guazzi et al. (2005) also measured brachial diameter in a subset of participants in the contralateral arm during and after hyperemia in tilt and found no changes; however, they combined healthy controls with those with pathophysiologic conditions such as hypertension and type 2 diabetes. Therefore, it remains unknown if the observed enhancement of vasodilation is local or systemic in healthy men and women. Indeed, Dietz et al. (1997) observed that during syncope, sympathetic withdrawal was not sufficient to explain the vasodilation that occurs, and that the vasodilation may act through mechanisms independent of β2-adrenergic and nitric oxide mediated mechanisms.

Rubini et al. (2012) conducted a study investigating the effect of the upright posture on the metabolic, cardiovascular, and electromyographic (EMG) activity of postural muscles (soleus and gastrocnemius) and they observed that the EMG activity of both muscles increased while upright. These results indicate increased muscle activation and presumably increased metabolite production while upright. However, it is important to note that they used a standing model rather than tilt table testing. We suggest that metabolites produced via upright posture (and potentially from deltoid and rotator cuff muscles from the elevated arm position of the imaged arm despite support) enter the ischemic forearm upon reperfusion causing an enhanced vasodilatory effect mediated by FMD related mechanisms such as adenosine and potentially lactate. Indeed, enhanced adenosine production is known to lead to syncope (Saadjian et al., 2002) and lactate stays in circulation for approximately 14 min (Almenoff et al., 1989). Hence, it is feasible that metabolites such as lactate stay in the general circulation long enough to enter the ischemic arm upon cuff release and possibly cause the increased non-shear stress dependent vasodilatory effect observed in our results. Systemic plasma concentrations of adenosine and lactate are needed during tilt table testing to determine their role in enhanced vasodilation.

Interestingly Guazzi et al. (2005) found a strong positive association between the change of HR during the tilt trial and the change of brachial artery diameter potentially linking peripheral vasodilation and the tachycardiac response to tilt. However, in the current study, even though MAP decreased and FMD increased during tilt, we did not observe a significant relationship between the changes in blood pressure and FMD. Contrary to our expectations we did not find evidence that women had enhanced FMD compared to men in either posture, yet they did have higher shear stress which is likely due to having smaller brachial arteries. Conflicting evidence about sex differences in the FMD response exist. Johns et al. (2020) suggested these discrepancies could be due to consideration of baseline brachial arterial diameter. For example, Juonala et al. (2008) found that women have greater FMD responses in comparison to men, however, after normalizing for baseline diameter, they found no significant differences in FMD between women and men and after similar normalization Shenouda et al. (2018) found lower FMD in women compared to men. Allometric scaling in the current study would not influence our primary results (i.e., change of FMD in upright posture) as there were no differences in baseline diameter in between postures. We also suggest that measurements of fitness or VO₂ should be conducted in future studies. In the current study, since the significantly higher shear stimulus in women compared to men did not elicit higher FMD in women, we suggest that our cohort of women have reduced endothelial function compared to our male cohort due to less times exercising per week leading to a lower estimated VO₂max. VO₂max has been shown to be correlated with endothelial health (Buscemi et al., 2013).

While the EndoPAT has been used to assess overall endothelial function, we used it as an index of assessment for β2-mediated dilation of the microvasculature, as our lab group previously observed that LnRHI is correlated with dobutamine induced vasodilation in the coronary artery (Nardone et al., 2020). We hypothesized that (1) LnRHI would be enhanced in the upright posture because of the increased β2 adrenergic receptor binding from sympathetic activation associated with the upright posture, and (2) in both postures, women would have higher LnRHI compared to men. Neither hypothesis was supported by our findings.

Goswami et al. (2013) investigated LnRHI and posture change in men and women previously and found no effect of sex or posture, however, they measured LnRHI after returning to the supine posture rather than while upright. For the current study, LnRHI was lower in the upright posture in both sexes suggesting reduced β2-mediated dilation in light of enhanced adrenergic responses to tilt. We speculate that prior to cuff release, the β2-receptors were saturated with norepinephrine which entered the ischemic arm via sympathetic nerve activity. We suggest that this was not observed as vasodilation prior to reperfusion (i.e., baseline brachial artery diameter did not change in the FMD trials when tilted) due to concurrent vasoconstrictor signals from α1-receptor stimulation. However, upon reperfusion and the associated stimuli, there was a reduced capacity for the β2-receptors to cause further dilation which would have been measured by LnRHI.

We did not observe a sex difference in resting LnRHI which was unexpected as Davis et al. (2020) found that women have greater Ln-RHI scores in comparison to age-matched men. This discrepancy could again be due to the underlying fitness differences of our participants. The men and women in Davis et al. (2020) were matched on the history of regular resistance exercise, and we did not control for fitness or physical activity. Hence, reduced fitness in women for the current study could have been a confounding variable responsible for attenuated vascular function.

Blood pressure fell over the course of the FMD trial, but not throughout the EndoPAT trial. This could have been due to any delays in the length of time that it took to find an adequate brachial artery image upon tilt (despite attempting to control for the time of cuff release). However, despite this fall in blood pressure, shear stress was not affected during reperfusion and FMD increased regardless. For the EndoPAT trials, the relatively higher (i.e., unchanged from baseline) blood pressure compared to the FMD trials could have been expected to increase LnRHI while upright, however, the opposite was observed.

We did not include measurements of muscle metabolism, plasma nitrates/nitrites, and oxidative stress markers, such as peroxynitrite, catecholamines, or other vasoactive substances. In order to test our hypothesis that the increased FMD in the upright posture is due to activation of postural muscles, future studies should consider measuring potentially vasoactive substances, such as lactate, adenosine, O₂ and CO₂, nitrates/nitrites, as well as electromyography of postural and shoulder muscles. Along with these measurements of muscle metabolism, we suggest conducting a true cardiopulmonary exercise test to determine fitness, as we used a VO₂ estimate via the Ainsworth equation. Lastly, we suggest measurements of alternate vascular beds (e.g., renal and splanchnic) to determine their role in the maintenance of blood pressure in light of the greater vasodilatory capacity in skeletal muscle beds while upright.

Our calculations of the relationships between the reduction of blood pressure of the course of the tilt FMD trial and the increase of FMD were underpowered. Greater sample numbers would help to strengthen any assumptions, which may be made using those data. Similarly, the statistically significant reduction of LnRHI in the upright posture was underpowered at β = 0.48; however, all other statistically significant comparison reached sufficient power of β > 0.8.