Influence of acute dietary nitrate supplementation timing on nitrate metabolism, central and peripheral blood pressure and exercise tolerance in young men

Twelve young healthy males [mean ± SD: age: 23 ± 4 years, stature: 1.80 ± 0.09 m, body mass: 75.8 ± 10.9 kg, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\text{O}}_{{{\text{2peak}}}}$$\end{document}V˙O2peak: 50.4 ± 8.5 ml^.kg^.min⁻¹, gas exchange threshold (GET): 118 ± 32 W, peak aerobic power (PAP): 323 ± 68 W] volunteered to participate in this study. None of the participants were tobacco smokers (Bailey et al. 2016) or vapers or taking any medication known to interfere with stomach acid production (e.g., proton pump inhibitors). No participants had any pre-existing medical conditions such as hypertension or diabetes. All participants were classified as recreationally active (McKay et al. 2022). Experimental testing was approved by Loughborough University Research Ethics Approvals Human Participants Sub Committee (ethics code: R18-P145) and confirmed with the principles of the Declaration of Helsinki, apart from registration in a database. Participants gave their written informed consent to participate.

Participants recorded their dietary intake 24 h prior to their first session and were asked to replicate this before subsequent visits. Each participant was given a list of NO₃⁻- and thiocyanate-rich foods (Dewhurst-Trigg et al. 2018) to abstain from eating 24 h before sessions and asked to avoid caffeine and alcohol ingestion in the 12 h and 24 h before each visit, respectively. All visits were conducted in a postprandial state. Since SFR is reduced in a state of hypohydration (Ship and Fischer 1997), participants were provided with 40 mL·kg⁻¹ body mass⁻¹ of fluid to consume in the 24 h before each visit (Minshull and James 2013) and instructed to consume 500 mL of water 1 h before testing to ensure euhydration on arrival. During testing sessions, participants were given 300 mL of water in 2 equal boluses to ensure euhydration was maintained. Since antibacterial mouthwash disrupts oral NO₃⁻ reduction (Govoni et al. 2008), participants were required to abstain from using mouthwash 48 h prior to each testing session. Participants were instructed to maintain their habitual exercise patterns for the duration of the study but were required to avoid strenuous exercise in the 24 h prior to each visit.

Participants reported to the laboratory on eight occasions. During the first visit, participants were familiarised with all the experimental procedures and completed a ramp incremental test for determination of GET, PAP and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\text{O}}_{{{\text{2peak}}}}$$\end{document}V˙O2peak. GET is a non-invasive estimate of the lactate threshold and demarcates the boundary between the moderate and heavy intensity exercise domains. PAP was the maximum power output attained during the incremental ramp test. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\text{O}}_{{{\text{2peak}}}}$$\end{document}V˙O2peak is defined as the highest volume of oxygen uptake during the incremental ramp test. During visit two, participants were familiarised with the time to exhaustion (TTE) exercise protocol. In the six main experimental visits, baseline measures of SFR and pH, BP and vascular function (pulse wave analysis) were obtained, in sequence, and salivary and plasma samples were collected for later assessment of [NO₃⁻] and [NO₂⁻]. Urine and serum osmolality were also evaluated at baseline to assess hydration status. Subsequently, participants ingested 2 × 70 mL of concentrated NO₃⁻-rich (BR; 13 mmol NO₃⁻) or NO₃⁻-depleted (PL; ~ 0.04 mmol NO₃⁻) beetroot juice (Beet It, James White Drinks Ltd., Ipswich, UK) with 30 g of cornflakes and 125 mL of semi-skimmed milk. Saliva measurements were repeated 1 h post beetroot ingestion. All baseline measurements were then repeated 2.5 h following beetroot ingestion to coincide with the peak plasma [NO₂⁻] (Wylie et al. 2013). Finally, participants completed the TTE exercise test. The six experimental conditions, PL and BR in the morning (started at 08:00; PL-MORN and BR-MORN), afternoon (started at 12:00; PL-AFT, BR-AFT) and evening (started at 15:00; PL-EVE and BR-EVE) were administered in a randomised, repeated-measures, crossover experimental design. PL and BR supplement administration was randomised (counterbalancing not possible due to the number of sequence permutations) and double-blinded (supplement bags labelled 1 and 2 by an independent investigator). Supplement ingestion occurred at 09:00, 13:00 and 16:00 in the MORN, AFT and EVE, respectively.

Statistical analysis was performed using IBM SPSS Statistics version 27. Shapiro Wilk’s test was used to check data normality. Baseline data and data containing one factor (condition [PL-MORN, BR-MORN, PL-AFT, BR-AFT, PL-EVE, BR-EVE] including hydration biomarkers, SFR, salivary pH, and TTE) were analyzed using one-way repeated-measures ANOVAs. Plasma [NO₃⁻] and [NO₂⁻], BP and vascular function were initially analysed using two-way repeated-measures ANOVAs (condition [PL-MORN, BR-MORN, PL-AFT, BR-AFT, PL-EVE, BR-EVE] × time [0 h and 2.5 h]). Salivary [NO₃⁻] and [NO₂⁻] were initially analyzed using two-way repeated-measures ANOVAs (condition [PL-MORN, BR-MORN, PL-AFT, BR-AFT, PL-EVE, BR-EVE] × time [0 h, 1 h, 2.5 h]). Significant ANOVA interaction effects were followed up with post hoc Dunnett’s tests for comparisons to baseline control for the salivary data and Holm-Bonferroni corrected paired-samples t tests were used for all other variables. To calculate effect sizes, partial eta squared (n_p²) was used for the omnibus tests and Cohen’s d_z (t/√n) for paired-samples t tests. All data are displayed as mean ± SD unless otherwise stated. Statistical significance was accepted at P ≤ 0.05.

For all participants, urine osmolality on arrival was < 700 mOsmol^.kg H₂O⁻¹ in all six conditions. Serum osmolality was between 285 and 295 mOsmol kg H₂O⁻¹ across all visits and not different between conditions (P > 0.050, n_p² = 0.03).

There were no inter-condition differences in salivary [NO₂⁻] at baseline (P > 0.050). There was a main effect for condition (P < 0.001, n_p² = 0.74) and time (P < 0.001, n_p² = 0.79), and a condition × time interaction effect (P < 0.001, n_p² = 0.66) for salivary [NO₂⁻]. Salivary [NO₂⁻] was unchanged between 0 and 2.5 h in PL-MORN, PL-AFT and PL-EVE (all P > 0.050). Salivary [NO₂⁻] was increased above baseline at all time points in BR-MORN, BR-AFT and BR-EVE (all P < 0.001), with no inter-condition differences at 1 h (d_z ≤ 0.16) or 2.5 h (d_z ≤ 0.42, both P > 0.050, Fig. 2). Normalising salivary [NO₂⁻] to SFR did not change any of the observed effects compared to absolute salivary [NO₂⁻].

Plasma [NO₂⁻] was not different between conditions at baseline (P > 0.050). There was a main effect for condition (P < 0.001, n_p² = 0.73) and time (P < 0.001, n_p² = 0.80), and a condition × time interaction effect (P < 0.001, n_p² = 0.76). Plasma [NO₂⁻] was unchanged between 0 and 2.5 h in PL-MORN (P > 0.050) but decreased in PL-AFT (P = 0.027) and PL-EVE (P = 0.050). Plasma [NO₂⁻] was increased above baseline 2.5 h post supplement ingestion in BR-MORN (642 ± 289 nM), BR-AFT (670 ± 314 nM) and BR-EVE (675 ± 355 nM) (all P < 0.001), with no differences between conditions (P > 0.050, d_z ≤ 0.11, Fig. 3).

Previous studies have shown both unstimulated SFR (Dawes 1975, 1972) and salivary pH (Choi et al. 2017; Ferguson and Fort 1974) exhibit circadian variability, being lowest during sleep and the early morning and peaking mid-afternoon. Contrary to previous findings, SFR and salivary pH did not exhibit a circadian rhythm in the current study with these variables not being significantly different across the morning, afternoon, and evening assessment points. It is well documented that food and fluid consumption can alter SFR and salivary pH (Ship and Fischer 1997; Brunstrom et al. 2000; Watanabe and Dawes 1988). Since fluid consumption and food intake was standardised over the 24 h preceding each testing session and participants were objectively determined to be euhydrated in the current study, the lack of diurnal variation on SFR and salivary pH is unlikely to be a result of altered dietary intake. However, it is plausible that our hydration protocol may have overridden the underlying daily rhythm in SFR. Moreover, both SFR and salivary pH were highly variable between-participants, which likely impeded the detection of any subtle changes in these variables across the day. We concede that a larger sample size may have been necessary to detect subtle changes in SFR and salivary pH. It should also be acknowledged that the methods used to collect and measure SFR and pH may not have been sufficiently sensitive to detect small diurnal variability in these responses. Previous studies have used passive drool techniques or fitted oral collection devices to collect saliva and have collected samples for 11–12 successive days between ~ 07:00 and 22:00 to evaluate SFR (Dawes 1975, 1972). Similarly, a previous study reporting circadian-like patterns in salivary pH captured continuous changes over 48 h using custom-made intraoral appliances (Choi et al. 2017). In contrast, the current study only measured SFR and salivary pH on nine occasions between 08:00 and 18:30 and with different techniques, which may account for the lack of a significant within-day variability in SFR or salivary pH herein.

Salivary and plasma [NO₃⁻] and [NO₂⁻] were not different between pre-supplementation baseline measures during the morning, afternoon, and evening experimental testing sessions. Consistent with previous research (Bailey et al. 2016; Cocksedge et al. 2023; Burleigh et al. 2018; Woessner et al. 2016), both salivary and plasma [NO₃⁻] and [NO₂⁻] increased following the acute ingestion of BR in the present study. However, contrary to the experimental hypothesis, the increases in salivary and plasma [NO₃⁻] and [NO₂⁻] after BR supplementation were consistent across the morning, afternoon, and evening. These findings align with previous research which has shown stability in plasma and urinary [NO₃⁻] (Ringqvist et al. 2000) and plasma [NO_x] (Tangphao et al. 1999) over a 24 h period. The lack of diurnal variation in the evaluated NO₃⁻ metabolism biomarkers in the current study may be partially attributed to the absence of a circadian rhythm in SFR and salivary pH. However, it is worth acknowledging that there may be diurnal variation in salivary and plasma [NO₃⁻] and [NO₂⁻] if assessed using more ecologically valid experimental designs (i.e., where individuals maintain their habitual dietary intake and activity levels) due to the NO₃⁻ content of the diet with different meals.

Despite evidence of circadian rhythms in BP reported in the literature (Douma and Gumz 2018; Williams et al. 2013; Jankowski et al. 2013; Boggia et al. 2016), no differences in baseline pre-supplementation brachial BP were observed between the morning, afternoon, and evening in healthy young men in the present study. Previous studies assessing 24 h BP have reported that the amplitudes of basal resting rhythms are small (3–6 mmHg peak-to-trough) in healthy young men and women (Scheer et al. 2010). Moreover, and also contrary to the experimental hypothesis, brachial SBP was not lowered in the morning, afternoon or evening after acute BR ingestion. Numerous previous studies have assessed the potential of NO₃⁻ supplementation to lower BP, with a lowering in brachial SBP after NO₃⁻ consumption observed in several (Bahadoran et al. 2017; Kapil et al. 2010; Larsen et al. 2006; Siervo et al. 2013; Bailey et al. 2010), but not all, previous studies (Siervo et al. 2013; Cermak et al. 2012; Zoughaib et al. 2023; Shepherd et al. 2015; Walker et al. 2019). However, a principal original contribution of the current study was evaluating the efficacy of NO₃⁻ supplementation to lower brachial BP at three different time points across the day and the observation that brachial SBP was not lowered in the morning, afternoon or evening after acute BR ingestion. The factors that regulate BP across the day are multifaceted (including but not limited to renal haemodynamics, the nervous system and mental/emotional stress) and highly complex (Smolensky et al. 2017), and as such it is unclear why reductions in brachial BP were not observed in the present study.

In contrast to the peripheral brachial artery SBP response, aortic SBP was lowered from the pre-supplementation baseline after BR supplementation by a similar magnitude in the morning, afternoon and evening. Lower aortic SBP after acute NO₃⁻ supplementation has been reported in some (Pekas et al. 2021; Kukadia et al. 2019; Hughes et al. 2016; Kim et al. 2019), but not all (Floyd et al. 2019) previous studies. In studies assessing both aortic and brachial SBP, a concurrent lowering in central and peripheral SBP has been reported (Pekas et al. 2021; Hughes et al. 2016; Kim et al. 2019), however, some studies have observed a reduction in central but not peripheral SBP (Kukadia et al. 2019; Mills et al. 2020). In contrast with the findings of the current study, both central and peripheral SBP have been reported to be lowered after acute NO₃⁻ supplementation in the morning (Pekas et al. 2021; Hughes et al. 2016; Kim et al. 2019), with studies reporting a greater effect on central than peripheral SBP not specifying the time of the day the assessments were completed (Kukadia et al. 2019; Mills et al. 2020). Both the lowering of central SBP and the increases in plasma [NO₂⁻] after BR ingestion were consistent across timepoints in the current study. Therefore, while previous studies have reported a strong agreement between plasma [NO₂⁻] and brachial SBP after acute NO₃⁻ supplementation, the current study suggests that central, but not brachial, SBP is modulated by circulating plasma [NO₂⁻]. The mechanisms for the lowering of brachial SBP after NO₃⁻ ingestion have been considered to be linked to the reduction of circulating plasma NO₂⁻ to NO leading to elevated cyclic guanosine monophosphate signalling leading to vasodilation (Kapil et al. 2020, 2010). However, NO₂⁻ can directly elicit vasodilation via s-nitrosylation (Bryan et al. 2005). In addition to increasing plasma [NO₂⁻], acute BR ingestion has been demonstrated to increase plasma S-nitrosothiol concentrations (Abu-Alghayth et al. 2021) which have been suggested to more closely reflect the improvement in vascular function after NO₃⁻ ingestion than plasma [NO₂⁻] (Pinheiro et al. 2015). It has also been suggested that the lowering in BP with elevated NO₂⁻ can occur via mechanisms independent of NO-cGMP signalling, and instead related to a novel, alternative redox pathway (mediated by hydrogen peroxide, persulfides and oxidation of protein kinase G1α), which culminates in NO-independent vasorelaxation (Feelisch et al. 2020). Therefore, further research is required to assess the mechanisms for the lowering in SBP after acute BR ingestion in humans and the extent to which this may differ in the control of central and peripheral SBP.

Acute BR ingestion largely did not modulate indices of arterial stiffness in the current study. Specifically, AP and AI were unaltered after BR ingestion in the morning or evening, but there was a small lowering in AP after BR ingestion in the afternoon. The clinical relevance of this finding in healthy young normotensive males is unclear (Wojciechowska et al. 2006). The general lack of improvement in pulse wave variables after acute NO₃⁻ ingestion is consistent with most (Pekas et al. 2021; Kim et al. 2019; Liu et al. 2013), but not all (Hughes et al. 2016), previous studies reporting the effects of BR on AP and AI in the morning. The current study extends these previous observations by assessing the effects of BR supplementation on these variables in the afternoon and evening. It is possible that chronic NO₃⁻ supplementation is required to improve pulse wave variables (Li et al. 2020) since arterial remodelling, including changes in the timing and/or magnitude of reflected waves from the peripheral arterial tree, may be necessary to elicit changes in central haemodynamics. Longer term NO₃⁻ supplementation and the potential for greater overall NO exposure could positively modulate endothelial homeostasis (Carlström et al. 2018), and vascular gene expression to support vascular function (Rammos et al. 2015). In turn, such effects could contribute to lower vascular resistance and associated pulse wave variables. Therefore, our findings suggest that acute BR ingestion is more likely to lower central versus peripheral SBP, and to not influence indices of arterial stiffness in healthy young males. This finding is consistent with research in pre-diabetic and diabetic individuals after daily ingestion of dietary NO₃⁻ for 6 months (Faconti et al. 2019). Although exact mechanisms of action are unclear, lowered SBP yet unaltered arterial stiffness with NO₃⁻ consumption has been postulated to be due in part to increased venodilation leading to decreased preload (Mills et al. 2020).

There were no changes in high-intensity cycling TTE between the morning, afternoon and evening timepoints after PL ingestion in the current study. This observation conflicts with previous studies reporting diurnal variation in maximal voluntary contractions (Chtourou et al. 2012; Martin et al. 1999), Wingate test performance (Chtourou et al. 2012, 2011; Hammouda et al. 2012; Lericollais et al. 2009; Souissi et al. 2004) and exhaustive severe-intensity cycling exercise (Hill 2014), with performance typically peaking in the afternoon and being lowest in the morning. Acute ingestion of BR providing ~ 13 mmol NO₃⁻ did not improve TTE during high-intensity cycling in the current study. While the existing literature generally supports a small but significant effect of NO₃⁻ supplementation to improve performance across a range of exercise settings (Senefeld et al. 2020), and acute NO₃⁻ supplementation has previously been reported to enhance performance during continuous high-intensity exercise tests (Wylie et al. 2013), there are also previous studies reporting no ergogenic effects in such settings (Cocksedge et al. 2020). It has previously been reported that sprint cycling performance is impaired in the morning compared to the afternoon and that acute BR ingestion (providing ~ 6.5 mmol NO₃⁻) can improve sprint cycling performance in the morning such that it is not different from the afternoon (Dumar et al. 2021). However, a limitation of that study was the lack of an appropriate placebo supplement to ensure the participants were blinded to the treatment conditions. The current study expands on this previous study by indicating that acute BR ingestion did not improve performance compared to PL in the morning, afternoon or evening. The lack of an ergogenic effect in the current study is unlikely to be due to an insufficient NO₃⁻ dose (Wylie et al. 2013). There is some evidence to suggest that NO₃⁻ supplementation may be more likely to improve continuous high-intensity exercise performance after multiple-day supplementation, due to the greater time course needed to increase muscle [NO₂⁻] (Kadach et al. 2023; Gilliard et al. 2018; Wylie et al. 2019) and subsequently impact skeletal muscle contractile function (Cermak et al. 2012; Jones et al. 2018), which may account for the lack of ergogenic effect of acute BR ingestion in the current study.

The findings of the current study suggest that acute BR supplementation is more likely to lower central SBP than brachial artery SBP across the course of the day. Whilst the measurement of brachial artery BP is well-established and provides strong clinical prognostic value, the importance of assessing central aortic BP and indices of aortic wave reflections has been clearly established in recent years (McEniery et al. 2014; Siervo et al. 2013). Indeed, the coronary arteries are exposed to central rather than peripheral pressures, which may account for observations that cardiovascular events may be more closely related to central pressures (McEniery et al. 2014). Moreover, the greater effect of BR ingestion on lowering central compared to brachial SBP is consistent with some studies reporting that central SBP is more likely to respond to antihypertensive treatments than brachial SBP (McEniery et al. 2014). The clinical relevance of the observed reductions in central SBP in healthy young men is currently unknown and warrants additional investigation. However, central SBP readings > 125 mmHg are associated with a significant increase in atherosclerotic cardiovascular outcomes, and for every 10 mmHg increase in central SBP the risk of an adverse cardiovascular outcomes increases by 11.7% (Kwon et al. 2022). This is important since resolving the time of day that administration of antihypertensive interventions elicits the optimal effects on cardiovascular health and cardioprotection remains unclear and an active area or research in cardiovascular medicine (Mackenzie et al. 2022; Hermida et al. 2010).

A challenge of administering NO₃⁻ acutely is that the second-pass metabolism of NO₃⁻ delays the attainment of peak plasma [NO₂⁻] until ~ 2–4 h post ingestion, and assessments of BP and exercise performance are recommended to take place upon attainment of peak plasma [NO₂⁻] (Wylie et al. 2013). To accommodate for the slow plasma [NO₂⁻] pharmacokinetics after NO₃⁻ ingestion, a limitation of the current study is that BP, vascular function and exercise performance measures were assessed early afternoon (~ 12:00–13:00), mid-afternoon (~ 16:00–17:00) and early evening (~ 19:00–20:00) after, respectively, ingesting BR in the morning (~ 09:00), early afternoon (~ 13:00) and mid-afternoon (~ 16:00). Therefore, the morning BP surge was not assessed in the current study, and the exercise test may not have been conducted early enough in the day to detect previously reported morning decrements in performance. Moreover, research in chronobiology has investigated the effect of chronotype; an individual’s predisposition towards morningness and eveningness, on responses to exercise (Vitale and Weydahl 2017). Studies have shown that circadian rhythms of physiological variables such as temperature are shifted dependent on chronotype characterisation, with biological rhythms in morningness types showing earlier peaks and troughs compared to eveningness types (Baehr et al. 2000; Bailey and Heitkemper 2001). Since chronotype was not characterized for the participants in the current study, this may have contributed to the observation of marked diurnal variability in exercise tolerance in the current study.