Cerebral blood velocity during concurrent supine cycling, lower body negative pressure, and head-up tilt challenges: implications for concussion rehabilitation

Introduction. The effect of concurrent head-up tilt and lower body negative pressure (LBNP) have been examined on middle cerebral artery velocity (MCAv) at rest; however, it is unknown the superimposed effect these factors have on blunting the elevation in cerebral blood velocity associated with moderate-intensity exercise. Methods. 23 healthy adults (11 females / 12 males, 20–33 years) completed three visits. The first consisted of a maximal ramp supine cycling test to identify the wattage associated with individualized maximal MCAv. Subsequent visits included randomized no LBNP (control) or LBNP at −40 Torr (experimental) with successively increasing head-up tilt stages of 0, 15, 30, and 45 degrees during the pre-described individualized wattage. Transcranial Doppler ultrasound was utilized to quantify MCAv. Two-factorial repeated measures analysis of variance with effect sizes were used to determine differences between days and tilt stages. Results. Between-day baseline values for MCAv, heart rate, and blood pressure displayed low variability with <5% variation. With no LBNP, MCAv was above baseline on average for all participants; however, 15 degrees and 30 degrees tilt with concurrent −40 Torr LBNP was sufficient to return MCAv to 100% of baseline values in females and males, respectively. Body-weight did not impact the association between tilt and pressure (R 2 range: 0.01–0.12). Conclusion. Combined LBNP and tilt were sufficient to reduce the increase in MCAv associated with moderate-intensity exercise. This exercise modality shows utility to enable individuals with a concussion to obtain the positive physiological adaptions associated with exercise while minimizing symptom exacerbation due to the notion of the Monro-Kellie doctrine.


Introduction
The brain is encapsulated by the skull, where the Monro-Kellie doctrine states the sum of brain parenchyma, cerebral spinal fluid, and cerebral blood flow (CBF) must remain constant to ensure intracranial pressure homeostasis (Monro 1783, Wilson 2016). However, following concussion, some individuals may experience an exacerbation of physiologically-based symptoms during physical exertion that is absent in healthy populations (Leddy et al 2007, O'Brien et al 2017, Graham et al 2021. This could potentially be attributed to deficits in autoregulatory and autonomic function, which could lead to a greater relative CBF increase during exercise, and further exacerbate these symptoms via the Monro-Kellie doctrine (Tan et al 2014). Therefore, developing novel techniques and exercise modalities that enable individuals with persisting post-concussion symptoms to obtain the positive benefits associated with exercise (e.g. elevated brain-derived neurotrophic factor, neural stem cell proliferation, decreased neuronal apoptosis) (Lucas et al 2015, Liu and Nusslock 2018, Gorski and De Bock 2019, Bliss et al 2020, Calverley et al 2020, while reducing symptom burden. This has been identified as a top research priority from the perspective of clinicians (Osmond et al 2023).
During exercise, a multitude of physiological changes occur; however, cerebral blood velocity (CBv; index of cerebral blood flow), generally follows a parabolic trend due to the change in chemical stimuli circulating within the cerebrovasculature (i.e. carbon dioxide) (Hellström et al 1996). CBv increases during mild to moderateintensity, before peaking at ∼60%-70% of an individual's maximal oxygen uptake. This coincides with an intensity slightly before an individual crosses into anaerobic metabolism at a respiratory exchange ratio of ∼1.0 (Beaver et al 1986). Here, the build-up of carbon dioxide is the greatest, which results in a ∼20% increase in CBv (Hellström et al 1996).
This elevation in CBv, in conjunction with impaired autoregulation and autonomic function, may underlie the increased symptomology of headache, pressure in the head, dizziness, and so forth with engagement in physical exertion Hovda 2001, 2014). This can nonetheless be blunted through external means (Yoshimoto et al 1994, Goswami et al 2018. For example, one way to artificially decrease CBv is through lower body negative pressure (LBNP), which redistributes blood flow into the venous system of the lower extremities (Crystal andSalem 2015, Goswami et al 2018). Pilot work from the author group using transcranial Doppler ultrasound demonstrated the~20% increase in cerebral blood velocity during cycling exercise can be attenuated with the application of LBNP (Miutz et al 2023). This data within ∼30 individuals demonstrated CBv was elevated compared to baseline at 0 and −20 Torr, slightly elevated at −40, and not different at −60 and −70 Torr levels of LBNP (Miutz et al 2023). Previous studies have additionally used tilt table methodological approaches to manipulate CBF, which utilizes gravity to shift blood from upper to lower body compartments (Alperin et al 2005,van Campen et al 2018, van Campen et al 2020. While both LBNP and tilt are possibilities to reduce CBF/ CBv, the combinatorial effect to attenuate the CBv increase during exercise has not been elucidated in the literature. A dose-response relationship has been proposed with respect to the aforementioned benefits of acute exercise (Herold et al 2019). Individuals with PPCS may experience transient symptoms with physical exertion, and thus creating techniques where they could exercise for slightly longer and/or at a higher intensity, may help improve recovery trajectories in those slow to recover from concussion. This would also enable them to obtain a greater extent of the systemic effects of exercise on the organs and cerebrovasculature (e.g. increased insulin sensitivity, reduced arterial stiffness, elevated mitochondrial oxidative capacity, etc) (Lucas et al 2015, Calverley et al 2020. Moreover, increases in intracranial pressure are thought to occur following concussion due to tissue swelling , which could additionally be blunted with external means. Therefore, this study aims to better understand the relationship between LBNP and tilt with supine cycling in a healthy population to determine the feasibility of employing this following concussion, which as stated is a top research priority identified by clinicians who treat concussion (Osmond et al 2023). The current protocol was first applied to a healthy population to lessen the burden of refining a methodological protocol in individuals with a concussion for safety and ethical reasons. A previous study by Tymko et al (2016), found CBv did not differ in steady-state conditions (without exercise) with 45 degrees head-up and head-down tilt independently; however, CBv was reduced when these were combined with −50 Torr LBNP. Therefore, it is hypothesized the combination of −40 Torr LBNP, and 45 degrees tilt would blunt the known ∼20% increase in CBv associated with submaximal exercise slightly below anaerobic threshold (Hellström et al 1996).

Ethical approval
The Conjoint Health Research Ethics Board at the University of Calgary (REB20-1662 and REB20-2112) provided the ethical approval for the current investigation. Prior to participation, participants provided written informed consent, researchers answered all questions, and thoroughly explained all protocols. Protocols were followed accordingly to institutional guidelines and standards set by the Declaration of Helsinki (aside from registering the study within a database).

Participants
Participants consisted of 23 healthy adults (11 females and 12 males) aged 20-33, who reported no diagnosis of concussion in the past 6 months. Participant demographics and environmental conditions are displayed in table 1. All participants were asked to complete the physical activity readiness questionnaire (PAR-Q+) (Warburton et al 2019), which requires participants to self-report any potential health complications/ contraindications that may arise during exercise. No participants recorded any complications and thus were cleared to engage in maximal exercise. Before all testing sessions, participants were asked to refrain from exercise, smoking, alcohol, vaping, and caffeine for 8 h prior to maintain consistency between testing days and ensure results were accurate (Burma et al 2020b, Burma et al 2020c. No participants were regular consumers of either tobacco or vaping products (Husten 2009).

Protocol
Participants completed three testing sessions within a week timespan, using a randomized crossover design (figure 1). The first visit consisted of a maximal ramp supine cycling test in order to identify the wattage that correlated with the peak CBv as previously described (Miutz et al 2023). This identified an individualized wattage associated with each participant's peak CBv to be used in the subsequent visits. Initial height and weight measurements were taken to determine each participant's stage increase in wattage during each minute of the exercise test until volitional fatigue. This wattage was calculated using the following formula (Miutz et al 2023), which has been demonstrated to elicit a robust parabolic MCAv response during supine cycling: Males: 0 .2 kilogram kg of body weight ( ) Females: 0 .15 kg of body weight.
As previously mentioned, peak MCAv is known to occur slightly before the anaerobic threshold. To minimize the potential impact of aerobic deconditioning and thus different energy systems contributing to the exercise condition (e.g. anaerobic versus aerobic metabolism), the second and third visits occurred within a 48-72 h time span. These consisted of two exercise sessions with progressively increasing head-up tilt that were completed in a randomized order. These were completed at the same time of day to minimize diurnal variation (Burma et al 2020a). During the second and third visits, 5 min of resting baseline data were collected. This was followed by 7 min exercise stages during successively increasing stages of tilt of 0, 15, 30, and 45 degrees during concurrent cycling (figures 1 and 2), for a total exercise duration of 28 min. The protocol for the two visits were identical, except one day was completed during −40 Torr of LBNP (experimental condition) while the other had no LBNP applied (control condition). During all stages of exercise, participants cycled at a cadence of 60-80 revolutions per minute (rpm).

Instrumentation
The LBNP chamber was custom-built with a supine cycle ergometer situated inside. This also contained a bicycle seat with adjusting pedal lengths. At the opening of the chamber, the participant was fitted with a wooden plank around the iliac crest and then secured around the waist with a plastic sleeve and neoprene bands in order to ensure a tight pressure seal. During all exercise tests, bilateral middle cerebral artery velocities (MCAv), heart rate, blood pressure, and end-tidal carbon dioxide (P ET CO 2 ) were monitored and collected. The MCAv was measured using transcranial Doppler ultrasound (TCD; DWL USA, Inc., San Juan Capistrano, CA, USA), where the velocity and depth were recorded for each participant during the first test and matched for the subsequent visits based upon depth and velocity. Two 2 MHz probes were placed at the transtemporal window using a headframe fitted to each participant (DWL USA, Inc., San Juan Capistrano, CA, USA). Carotid compressions were used to ensure the artery insonated was fed from the internal carotid artery (Willie et al 2014). Heart rate was monitored using a Polar heart rate monitor fitted using a chest strap (Polar H10, Kempele, Finland). Additional measures of heart rate were collected using a 3-lead ECG (FE 231 BioAmp; AD Instruments, Colorado Springs, CO, USA). Finger photoplethysmography was used to collect blood pressure across the cardiac cycle and was corrected at heart level (Finometer NOVA; Finapres Medical Systems, Amsterdam, The Netherlands) (Omboni et al 1993, Sammons et al 2007. P ET CO 2 was collected through capnography with an inline gas analyzer (ML206; AD Instruments). All data were collected using LabChart and stored offline analysis with this commercially available software (LabChart Pro Version 8, AD Instruments).

Data processing
Bilateral MCAv's were taken as a precautionary measure in case one of the two probes experienced movement error during exercise. Therefore, for the final measure of CBv, the stronger of the two MCAv's was used to ensure the conclusions were based upon physiological differences between conditions, rather than measurement/ movement and/or sonographer error. However, the within-participant comparisons were completed from the MCAv on the same side of the head. During the maximal exercise test, the last 40 s of each minute-long stage were averaged to determine MCAv. During the second and third sessions, the last 3 min of the 7 min stages were used to determine the average MCAv for each stage. Previous work has noted that supine-to-stand orthostatic fluid shifts generally stabilize by ∼60 s in the majority of individuals (Harms et al 2020). As a tilt change of 15 degrees is substantially less than a 90 degrees change from supine-to-standing, the initial 4 min buffer at the start of each stage ensured participants reached a steady-state following the fluid shift associated with each tilt stage. While absolute MCAv were recorded, the relative metric for each stage was calculated as the percent change compared to the baseline average, as seen below.
Sample size calculation A sample size calculation was completed using G * Power (Version 3.1.9.6) based on the findings from Tymko and colleagues (2016), who compared MCAv responses during 5 min of −50 Torr LBNP within three positions: head-up 45 degree tilt, head-down 45 degree tilt, and supine. These authors noted a 15 cm s −1 reduction in MCAv when comparing between the supine baseline and after 5 min LBNP within the head-up tilt position, which is nearly identical to the stimulus used in the current investigation. This magnitude difference led to an f effect size of 0.91, which with an alpha and beta of 0.05 and 0.80, respectively, produced a required sample size of 10 participants. However, previous research has identified orthostatic intolerance differences between males and females (Cheng et al 2011). Therefore, the sample was doubled to enable a comprehensive comparison between LBNP and no LBNP, while also exploring the potential confounding influence sex has on the MCAv response during the experimental protocols.

Statistical analysis
All statistical analyses were performed using R-Studio (Version 2022.7.1.554) (R Core Team 2020). Differences between days and stages for absolute and relative MCAv were assessed using a two-factorial repeated measures analysis of variance (ANOVA): days (pressure and no pressure), stage (baseline, 0 degrees, 15 degrees, 30 degrees, and 45 degrees tilt), and the interaction between days and stages (Blanca et al 2017). Levene's tests were run to determine if any comparisons violated the homogeneity assumption between groups. Tukey's post-hoc pairwise comparisons were conducted to assess differences between groups in the case of a significant omnibus test. Sex comparisons at each stage were assessed using Wilcoxon rank sum tests. Based on literature suggesting making inferences based upon a binary p-value may not be the most appropriate for biomedical/physiological literature, inferences were made based on p-values and the associated effect size (Amrhein et al 2019, Panagiotakos 2008, Halsey 2019, Bakeman 2005. For the ANOVAs, generalized eta squared (η 2 G ) coefficients were used with thresholds of <0.02 (negligible), 0.02-0.14 (small), 0.14-0.26 (moderate), and >0.26 (large) (Bakeman 2005). Cohen's d coefficients were used for the Tukey post-hoc comparisons with thresholds of <0.20 (negligible), small (0.20-0.50), moderate (0.50-0.80), and large (>0.80) were used (Lakens 2013). Wilcoxon effect sizes (r) were used for the sex comparisons with thresholds of: <0.10 (negligible), 0.10-0.30 (small), 0.30-0.50 (moderate), and >0.50 (large) (Maher et al 2013). To understand if body weight confounded the outcome metrics of interest, basic linear regressions (R 2 ) were completed for all stages of tilt across both exercise conditions. These were conducted on the relative MCAv metrics and stratified by biological sex, as lower body weight has been shown to result in a greater predisposition to orthostatic intolerance (Rutan et al 1992). Females and males differ in body weight and mass, and therefore these regressions were stratified by sex to control for both potential modifying factors of sex and body weight. While R 2 estimates have no agreed-upon thresholds as these will vary depending upon the field of study, a priori cut-offs consistent with previous literature were used: <0.10 (negligible), 0.10-0.30 (small), 0.30-0.50 (moderate), 0.50-0.80 (large), and 0.80-1.00 (very large) (Burma et al 2022). Variability between baseline physiological variables was assessed through coefficient of variation (CoV) estimates (Hopkins 2000). Alpha was set a priori at 0.05.

Physiological parameters
All cardiorespiratory, cerebrovascular, and cardiovascular parameters are displayed in figure 3 across both days and all tilt stages. Baseline values between days are shown in table 2, with excellent reliability as displayed through the low coefficient of variation values (all <8.0%) and the MCAv being noted at <3.0%.

Absolute CBv
Day (F (1,220) = 3.90, p = 0.049, η 2 G = 0.02 [small]) and stage (F (4,220) = 3.31, p = 0.012, η 2 G = 0.06 [small]) main effects were significant for absolute MCAv metrics, whereas the stage-by-day interaction was not (F (4,220) = 0.51, In contrast to the absolute differences, the only relative sex difference was noted during the pressure day at a tilt of 15 degrees (p = 0.032, r = 0.42 [moderate]), where females had lower MCAv values (figure 7). All other comparisons showed no significance (p > 0.079, r 0.38 [small-to-moderate]) (figure 7). Figure 8 displays the influence body weight has on the relative MCAv change from baseline for each participant at each stage, stratified by sex. Weight appeared to play a minor role during no pressure for females (R 2 range: 0.22-0.44); however, weight was inconsequential for males (R 2 range: 0.01-0.09) (figure 8). During the pressure condition, weight had a minimal impact for both sexes (female R 2 range: 0.02-0.12; male R 2 range: 0.01-0.03) (figure 8). However, with pressure, the slope was the smallest and closest to baseline MCAv (100%) at 15 degrees and 30 degrees tilt for females and males, respectively (figure 8). . Average with 95% confidence intervals for all cardiovascular and respiratory parameters during each stage of tilt in 23 total participants (12 females and 11 males) during lower body negative pressure (LBNP) and no LBNP conditions. Beats per minute (bpm), breaths per minute (brpm), millimeters of mercury (mmHg). Table 2. Cerebrovascular, cardiovascular, and respiratory parameters across both days and each stage of tilt, stratified by sex (23 total: 12 females and 11 males). The coefficient of variation (CoV) was calculated to determine the between-day variability for baseline values. This ensured any differences were due to the lower body negative pressure (LBNP) stimulus and not natural between-day variations.

Discussion
This study examined the superimposed effect of head-up tilt and LBNP during supine cycling specifically on its ability to blunt the ∼20% increase in CBv associated with moderate-intensity exercise (Hellström et al 1996).
The main findings from the study were: (1) without concurrent −40 Torr LBNP, MCAv remained above baseline values for all stages of tilt for males and females; (2) during the pressure condition, MCAv increased above baseline at 0 degrees tilt and returned to baseline at 15 degrees tilt for females and 30 degrees for males; (3) females continued to fall below baseline values at 30 and 45 degrees tilt, with males followed a similar trend at 45 degrees tilt, (4) body mass did not modify the superimposed effect of tilt and LBNP, increasing the generalizability of the findings. Collectively, these findings highlight that concurrent LBNP and head-up tilt, have a combined effect to further decrease MCAv values compared to the independent effect of these techniques.  . This is consistent with the findings in the present investigation, albeit the current study noted MCAv slightly decreases at each stage of tilt without pressure (figures 4 and 7). Nonetheless, this remained elevated compared to baseline for males and females, even at 45 degrees ( figure 7). An important difference between studies is the fact participants completed concurrent supine exercise in the current investigation, while participants within Tymko et al (2016), were under 'resting' conditions. Another study by Deegan et al (2010), examined the combined effect of head-up tilt and LBNP, using . Absolute and relative changes in middle cerebral artery (MCA) velocity across all stages in 23 total participants (12 females and 11 males) during lower body negative pressure (LBNP) and no LBNP conditions. Two-factorial repeated measures analysis of variance were used to determine differences between days and tilt stages. The Phi symbol (Φ) denotes a stage that differed compared to baseline. The Psi symbol (Ψ) denotes a stage that differed compared to 0 degrees tilt. The Upsilon symbol (ϒ) denotes a stage that differed compared to 15 degrees tilt. The Sigma symbol (Σ) denotes a stage that differed compared to 30 degrees tilt. Centimetres per second (cm/s), percent (%).

Comparison with previous research
70 degree tilt and progressive negative pressure until presyncope symptoms emerged. These authors noted MCAv reduced by ∼40% by the end of their protocol (Deegan et al 2010), compared to the ∼10% reduction seen within the current investigation. Two large differences between studies are participants in the current protocol concurrently exercised, which caused the slight elevation in MCAv, as well as the LBNP stimulus was only −40 Torr, compared to a maximum of −80 Torr in the study by Deegan and colleagues (2010). Therefore, while slight differences in methodological designs, it appears the current results are physiologically similar to other investigations combining LBNP and tilt (  It is well-known females have a higher resting CBv compared to males (Martin et al 1994, Alwatban et al 2021; however, this dissipates when normalized according to each individual's baseline ( figure 7). Interestingly, the only relative MCAv sex difference in the current investigation was at 15 degrees tilt during the pressure day, with females having a reduced value compared to males that disappeared at 30 degrees (figure 5). Panel D in figure 5 highlights the discrepancies are due to female's MCAv returning to baseline at 15 degrees tilt with −40 Torr, while males remained slightly elevated. However, at 30 degrees tilt, male's MCAv was near ∼100% of baseline (figure 5(D)) denoting this stimulus was sufficient to attenuate the ∼20% increase in CBv associated with a respiratory exchange ratio of ∼1.0 (Beaver et al 1986, Hellström et al 1996. Moreover, when controlling for body weight stratified by sex, figure 8 highlights the regression line was nearly horizontal around 100% of baseline with an R 2 value closest to zero at 15 degrees and 30 degrees tilt for females and males, respectively. This difference between sexes is in agreement with previous reports noting females have a slightly greater degree of orthostatic intolerance, which may stem from several proposed physiological factors (e.g. higher estrogen levels, more active sympathetic system, etc) (Cheng et al 2011). Interestingly, figure 8 highlights body weight explained a degree of the variance in females when examining all stages with no LBNP applied; however, no association was found for males. Further investigation into this finding is warranted. Other investigations utilizing progressive LBNP to compare between sexes noted minimal differences between males and females regarding the decrease in CBv (Rosenberg et al 2021). The current study also found body weight explained a degree of variance for females during exercise and tilt without LBNP applied, which is consistent with previous literature stating an inverse correlation exists between body weight and orthostatic intolerance (Rutan et al 1992).

Implications for concussion rehabilitation
Findings from this study may have paramount implications for post-concussion recovery and novel treatment strategies. While it is known engaging in exercise reduces days to symptom resolution across all phases of concussion recovery, some individuals may experience an exacerbation of symptoms due to physical exertion . This ultimately would limit their ability to obtain the positive physiological benefits of exercise (    A potential explanation for this could be the result of impaired autoregulatory capabilities and a disruption of the Monro-Kellie doctrine (Monro 1783, Wilson 2016). This would result in elevated intracranial pressure, greater pressure on the meninges, and may partially explain why previous reports have noted the common symptoms associated with physical exertion are headaches, pressure in the head, and dizziness (Graham et al 2021).
The current study presents a possibility for future exercise regimens as a treatment option for those who experience post-concussion exercise intolerance due to physiological disruptions (Leddy et al 2007), as they could engage in an exercise modality that abolishes the elevation in CBv associated with moderate-intensity exercise (Miutz et al 2023). Therefore, these individuals would be able to engage in exercise for a longer duration and/or at a higher intensity, enabling greater exercise-induced physiological benefits (e.g. angiogenesis, neurogenesis, elevated brain-derived neurotrophic factor, regeneration of nerve cells, etc) (Lucas et al 2015, Liu and Nusslock 2018, Gorski and De Bock 2019, Bliss et al 2020, Calverley et al 2020. Nonetheless, further work is warranted to confirm this proposition, especially considering it appears those with a concussion may respond in a slightly different manner to LBNP compared to healthy controls (Worley et al 2021). For example, Worley and colleagues (2021) noted that MCAv responded differently following concussion; however, these differences occurred during lower body positive pressure rather than LBNP. Therefore, while the current findings show promise, further work is warranted to solidify the current findings following concussion when both exercise and tilt are added to LBNP. Finally, this additional may be influenced by the severity of the brain injury, where a greater stimulus is required for those with a severe traumatic brain injury compared to someone with a mild or moderate injury (Ding et al 2020).
Individual variability may make it difficult to prescribe one type of exercise regimen, especially given the fact concussion is a very heterogeneous injury (known as a 'snowflake' injury), where a multifaceted treatment approach is warranted (Schneider 2019). Therefore, the degree of tilt and/or LBNP may need to be adjusted on an individualized basis to blunt the exercise-induced elevation in MCAv. For example, figures 7 and 8 shows the average MCAv returned to baseline during −40 Torr LBNP at 15 degrees tilt for females and 30 degrees for males. However, there were some individuals still above baseline at 45 degrees tilt, while others fell below baseline during −40 Torr LBNP at 0 degrees tilt. This means, the specific pressure and/or tilt may need to be slightly adjusted based on individual physiology; however, the current results demonstrate a very good starting point for these individualized exercise prescriptions to be developed from. As such, these values provide a foundation, which can be modified during testing to ensure an individual's MCAv is at or below.
Another factor to consider is the noise produced by the equipment used to create the LBNP (Tymko 2016). However, figure 2 demonstrates this equipment was contained within sound-dampening foam, to minimize the extent this would cause participants to report an elevation in their 'sensitivity to noise'. Moreover, this would additionally be minimized if participants are provided with earplugs to further dampen the sound. Increasing LBNP is accompanied by an increase in sound volume, hence using the lowest possible LBNP stimulus is beneficial to prevent the aggravation of symptoms unnecessarily. This stresses the importance of the current findings denoting the benefits of combining tilt and LBNP.
Limitations TCD uses ultrasound waves to measure the velocity of red blood cells, which has great temporal resolution and low sensitivity to movement artifacts (Purkayastha and Sorond 2012). This enables it to be used during exercise protocols while producing robust results. However, the caveat of the TCD is that it assumes the diameter of the vessel remains constant to provide a surrogate for CBF (Ainslie and Hoiland 2014). Given exercise is known to elevate blood pressure and circulating carbon dioxide levels (Ogoh and Ainslie 2009), it is likely diameter may have had subtle changes. However, the two exercise tests were similar with respect to P ET CO 2 and respiration patterns, so it is unlikely this affected the MCAv outcome measures. Further, it should be noted an extra control condition of 28 min of exercise in the 45 degree tilt position was not included in the current investigation. This is due to the fact that this protocol with consistent tilt and no LBNP would result in a steady-state MCAv as the exercising workload would be below anaerobic metabolism. Hence, the MCAv response that would occur would likely be representative of the MCAv seen during the last 7 min of the control condition with no LBNP applied at 45 degrees and throughout the upright cycling literature (Smith and Ainslie 2017, Burma et al 2020c, Burma et al 2020b. This mitigates the concern of an extra 28 min control condition providing additional burden to participants. Menstrual cycle, hormones, and contraceptive usage were not tracked among female participants. Nonetheless, pilot work from the author group has demonstrated the orthostatic challenge applied in the current investigation is minimally influenced by the menstrual cycle phase. Previous work has noted autoregulatory capabilities are not different between younger and older populations (Smirl et al 2015, Carey et al 2000, Maxwell et al 2022; however, orthostatic intolerance is known to become more prevalent with increasing age (Rutan et al 1992). Therefore, the current study warrants further exploration across adulthood and within the elderly. A final limitation is that cardiorespiratory fitness status for each participant was not measured in the current investigation, as previous work has noted cerebral autoregulation is impacted by this variable (Labrecque et al 2017. Nevertheless, the study design of the current investigation was a randomized crossover design where each participant acted as their own control, which controls for any covariates that would impact the outcome measures (Mills et al 2009).

Conclusion
This study aimed to elucidate the effect of combined LBNP and tilt on CBv during supine cycling. Overall results show a combined effect of −40 Torr LBNP with 15 degrees head-up tilt for females and −40 Torr LBNP with 30 degrees head-up tilt for males resulted in an abolishment of the~20% elevation in CBv associated with moderate-intensity exercise (Hellström et al 1996). Conversely, without applied negative pressure, CBv on average remained elevated even at the highest degree of tilt investigated (45 degree). Following concussion, some individuals are known to experience symptom exacerbation during physical exertion due to underlying disruptions to their cerebrovascular physiology. This may be associated with an increase in CBv and an elevation in intracranial pressure due the Monro-Kellie doctrine. These current findings show great promise/utility within future concussion rehabilitation as this may provide a novel technique for individuals who are limited in their exercise capacity due to symptom exacerbation associated with physical exertion to overcome this barrier. Furthermore, this may also impact individuals who experience persistent post-concussive symptoms and may allow them to obtain the positive benefits of exercise (e.g. brain-derived neurotrophic factor, neural stem cell proliferation, neurogenesis, etc) to a greater extent compared to typically cycling and/or treadmill exercise. Further work within a symptomatic concussion population is warranted to confirm these propositions.