The primary aims of this investigation were to (1) test the hypothesis that post-exercise brachial artery FMD would be attenuated in a dose-dependent manner by varying the dose of retrograde shear in the brachial artery during lower body exercise, and (2) test the hypothesis that vitamin C supplementation would prevent the attenuation of brachial artery FMD following elevated brachial artery oscillatory and retrograde shear during lower body exercise. The primary findings indicate that (1) brachial artery FMD is attenuated following exercise with oscillatory and retrograde shear above the normal response, but the attenuation is not different across the applied gradations of retrograde shear, and (2) vitamin C supplementation prevents the decline of FMD induced by augmented oscillatory and retrograde shear during exercise.
We did not observe a graded effect of cuff pressure on post-exercise FMD, despite achieving statistically different SR between the lowest and highest cuff pressures. In other words, the overall goal of applying different shear between exercise sessions was achieved but was not reflected in graded effects on post-exercise FMD. The lack of a dose effect on FMD is therefore interpretable as a true negative.
In a recent review, it was postulated that increased antegrade shear during exercise provides a beneficial stimulus to the endothelium which may counteract the possible negative influence of concurrent augmented retrograde shear . The findings from the present study support this hypothesis. The greatest dose of retrograde shear did not further impair post-exercise FMD beyond what was observed for the lowest dose of retrograde shear. These results are in contrast to those obtained from augmented retrograde shear during rest and therefore refute our original hypothesis that FMD would be attenuated in a dose-dependent manner by various doses of retrograde shear in the brachial artery during lower body exercise. Thijssen et al. observed a dose–response relationship between brachial artery retrograde SR and brachial artery FMD at rest without a concurrent increase of antegrade shear . Given these contrasting results, it appears that augmented antegrade shear associated with exercise partially ameliorates the deleterious effects of elevated oscillatory and retrograde shear on post-exercise endothelial function.
Augmented antegrade SR during exercise may reduce the impact of retrograde shear on post-exercise FMD by limiting the increase of the oscillatory pattern. During exercise in all conditions, the OSI was increased and the control arm was lower than the cuffed but there were no differences between cuff pressures. Therefore, the amount of oscillation during exercise appears to at least partially influence post-exercise FMD. This is supported by the lack of a difference of OSI between cuffed arms which resulted in post-exercise FMD that were equivalent. Tinken and colleagues performed two studies where brachial artery SR profiles were modified during handgrip exercise  and lower body cycling  using forearm compression. Although OSI was not calculated in either study, it appeared to increase in the cuffed arm in both investigations, primarily due to significantly lower antegrade shear as retrograde SR was unchanged during the interventions. These shear patterns resulted in post-exercise FMD that were either unaltered  or decreased from baseline . In the present study, despite the increase in antegrade SR during exercise, slightly elevated OSI and retrograde SR during exercise attenuated post-exercise FMD. This indicates that even minimal increases in oscillatory and retrograde shear during exercise can substantially influence post-exercise endothelial function. Previous results [7, 23] and those from the present study indicate that even minimal increases in retrograde SR and OSI above the normal response during exercise may play a prominent role restricting or reducing post-exercise FMD.
It is challenging to differentiate which component of the shear profile (i.e. antegrade, retrograde, mean, or OSI) during exercise provides the appropriate stimulus for improving post-exercise endothelial function. OSI, antegrade, and retrograde SR were elevated during all exercise conditions in the control arm which increased post-exercise FMD. Further augmentation of oscillatory and retrograde SR in the cuffed arm resulted in an attenuated post-exercise FMD. Tinken et al.  found an augmented post-exercise brachial artery FMD following handgrip exercise which increased antegrade, retrograde, mean shear, and presumably OSI. However, mean shear in the control arms of the present investigation did not increase during the 60 mm Hg sessions, yet post-exercise FMD was still augmented. Collectively, it appears as though exercise in these non-cuffed conditions elicited appropriate antegrade, retrograde and oscillatory shear responses which augmented post-exercise FMD.
The findings from this investigation demonstrate that vitamin C prevents an attenuated post-exercise FMD following elevated oscillatory and retrograde shear during exercise. The oscillatory and retrograde shear patterns induced by cuff pressure in the placebo condition likely produced reactive oxygen species which overcame local antioxidant defenses. Antioxidant status in the control arm appears to maintain or lower oxidative stress in response to the normal shear patterns induced by the exercise bout which improved FMD. Retrograde and oscillatory shear enhance reactive oxygen species production in cultured endothelial cells by increasing NADPH oxidase subunit mRNA expression and augmenting subunit phosphorylation [11, 12], increasing production of reactive oxygen species by xanthine oxidase  and mitochondria  which leads to the oxidation of BH4 to BH3· radicals [14, 32]. Low BH4 bioavailability uncouples eNOS thereby reducing eNOS function and augmenting eNOS superoxide production [32, 33]. Vitamin C supplementation appears to preserve post-exercise NO bioavailability in response to augmented oscillatory and retrograde shear during exercise. Since vitamin C was only administered prior to the highest dose of retrograde shear we cannot explicitly state that oxidative stress contributes similarly to lower retrograde shear conditions. Beyond the antioxidant effects of vitamin C, it appears to reduce BH3· radicals back to BH4, which would improve eNOS production of NO .
Our study has several limitations. FMD data following exercise should be interpreted with caution due to several vascular alterations (i.e. basal arterial diameter, sympathetic tone, blood flow and shear stress, and reactive hyperemia) which potentially contribute to the results . In particular, significantly greater post-exercise baseline diameters in the cuffed arm may have artificially confounded the FMD results due to the mathematical bias against large baseline diameters. However, we did assess FMD as the absolute change from baseline to post-occlusion peak diameter and found identical results (data not shown). Also, smooth muscle function was not assessed before or after exercise; however manipulated SR patterns during exercise using similar experimental procedures do not appear to alter smooth muscle function [7, 23]. Notably, a circulating marker of oxidative stress was not measured to confirm a reduction in reactive oxygen species. Currently, there is no clear consensus for the optimal circulating biomarker of intracellular oxidative stress, and the relevance of measuring a circulating marker as an indication of the oxidative state within a cell may be questionable. Since we did not measure a marker of oxidative stress, we cannot clearly state that post-exercise FMD following vitamin C supplementation was preserved by a decreased oxidative stress. However, observations indicate that augmented oxidative stress is abolished when vitamin C is given prior to several different types of interventions [37–39]. Furthermore, post-exercise FMD in the cuffed arm with prior vitamin C supplementation was similar to the control arm in the placebo and vitamin C conditions, which indicates that NO bioavailability was unaffected. Finally, diet was not strictly controlled with our subjects; therefore dietary antioxidants may have varied between exercise sessions. To reduce this possible variation between sessions, our subjects fasted according to the latest dietary guidelines for FMD assessments  and were asked to maintain their normal dietary habits throughout the study.