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

Purpose Dietary nitrate (NO3−) supplementation can lower systolic blood pressure (SBP) and improve exercise performance. Salivary flow rate (SFR) and pH are key determinants of oral NO3− reduction and purported to peak in the afternoon. We tested the hypotheses that NO3−-rich beetroot juice (BR) would increase plasma [nitrite] ([NO2−]), lower SBP and improve exercise performance to a greater extent in the afternoon (AFT) compared to the morning (MORN) and evening (EVE). Method Twelve males completed six experimental visits in a repeated-measures, crossover design. NO3−-depleted beetroot juice (PL) or BR (~ 13 mmol NO3−) were ingested in the MORN, AFT and EVE. SFR and pH, salivary and plasma [NO3−] and [NO2−], brachial SBP and central SBP were measured pre and post supplementation. A severe-intensity exercise tolerance test was completed to determine cycling time to exhaustion (TTE). Results There were no between-condition differences in mean SFR or salivary pH. The elevation in plasma [NO2−] after BR ingestion was not different between BR-MORN, BR-AFT and BR-EVE. Brachial SBP was unchanged following BR supplementation in all conditions. Central SBP was reduced in BR-MORN (− 3 ± 4 mmHg), BR-AFT (− 4 ± 3 mmHg), and BR-EVE (− 2 ± 3 mmHg), with no differences between timepoints. TTE was not different between BR and PL at any timepoint. Conclusion Acute BR supplementation was ineffective at improving TTE and brachial SBP and similarly effective at increasing plasma [NO2−] and lowering central SBP across the day, which may have implications for informing NO3− supplementation strategies.


Introduction
Diurnal variation in acute cardiovascular events is well established, with epidemiological data revealing increased incidence of strokes (Elliot 1998;Sheppard et al. 2015), myocardial infarctions (Cohen et al. 1997;Fabbian et al. 2017), and sudden cardiac death (Cohen et al. 1997) in the morning.Typical morning behaviours, including arousal from sleep, sudden postural changes, increased activity and psychological stress, instigate increases in sympathetic tone, vasoconstriction and peripheral arterial resistance, which contribute to the 'morning surge' in blood pressure (BP) (Kario 2010).Both peripheral and central BP exhibit parallel circadian rhythms, with lower values manifesting during night-time sleep, followed by abrupt increases with morning wakening before attaining peak values in the late afternoon (Douma and Gumz 2018).Diurnal fluctuations in exercise performance across a range of sport and exercise settings is also well established, with performance purported to attain peak and nadir levels in the afternoon and morning, respectively (Chtourou et al. 2011(Chtourou et al. , 2012;;Hammouda et al. 2012;Lericollais et al. 2009;Martin et al. 1999;Souissi et al. 2004;Hill 2014).Although BP and exercise performance exhibit a diurnal variation, the efficacy of dietary interventions to modulate these diurnal responses is unclear.
Dietary nitrate (NO 3 − ) supplementation has been reported to improve various aspects of cardiovascular function, including lowering resting brachial BP and arterial stiffness variables, and to improve exercise performance (Jackson et al. 2018;Bahrami et al. 2021;Li et al. 2020;Senefeld et al. 2020).These effects have been linked to increased circulating plasma [nitrite] ([NO 2 − ]), a substrate for nitric oxide (NO) production via the so-called NO 3 − -NO 2 − -NO pathway (Kapil et al. 2020).After ingestion, approximately 25% of NO 3 − enters the enterosalivary circulation, being absorbed by, and concentrated in, the salivary glands on the first-pass (Govoni et al. 2008).Subsequently, NO 3 − -rich saliva is secreted into the oral cavity wherein NO 3 − undergoes second-pass metabolism by anaerobic bacteria on the tongue which reduce salivary NO 3 − to NO 2 − (Doel et al. 2005;Duncan et al. 1995).Once swallowed, a portion of this NO 2 − is reduced to NO and other reactive nitrogen intermediates in the stomach (Benjamin et al. 1994), with some NO 2 − and reactive nitrogen intermediates entering systemic circulation for later NO generation (Kapil et al. 2020).Whilst NO 3 − supplementation has the potential to improve BP, vascular function, and exercise performance, it is currently unclear whether such effects are consistent across the day.
The efficacy of the NO 3 − -NO 2 − -NO pathway to elicit physiological effects is dependent on NO 3 − transport into the oral cavity and the host oral microbiome for NO 3 − reduction (Govoni et al. 2008;Bailey et al. 2016;Hezel and Weitzberg 2015;Jansson et al. 2008;Lundberg 2012).Indeed, when salivary NO 3 − uptake and secretion or oral NO 3 − reduction are impaired, the increase in plasma [NO 2 − ] and lowering in BP after NO 3 − supplementation are attenuated (Govoni et al. 2008;Bailey et al. 2016;McDonagh et al. 2015).On the other hand, secretion of NO 3 − into the oral cavity and exposure to the oral NO 3 − -reducing anaerobes will be enhanced by increasing salivary flow rate (SFR).However, although previous research has shown that music stimuli can elevate saliva secretion and salivary NO 2 − generation (Jin et al. 2018), it is presently unclear whether diurnal variation in SFR impacts salivary and plasma [NO 3 − ] and [NO 2 − ].In addition to SFR, salivary pH can impact oral NO 3 − reduction.Specifically, increasing salivary pH after NO 3 − supplementation has been reported to increase salivary and plasma [NO 2 − ] ( Cocksedge et al. 2023).Since both unstimulated SFR (Dawes 1975(Dawes , 1972) ) and salivary pH (Choi et al. 2017;Ferguson and Fort 1974) exhibit circadian rhythms, with an acrophase in the afternoon, oral NO 3 − reduction and the resultant increases in salivary and plasma [NO 2 − ] after NO 3 − supplementation may be greatest in the afternoon.Consistent with this postulate, oral NO 3 − reduction was reported to be enhanced in the afternoon compared with the morning during a mouth rinse with a KNO 3 − solution (Rowland et al. 2021).However, it is unclear whether enhanced oral NO 3 − reduction in the afternoon during a brief mouth rinse is reproducible after NO 3 − ingestion and whether this effect translates into greater plasma [NO 2 − ], and more pronounced reductions in BP and improvements in exercise tolerance.
The purpose of this study was to investigate the effect of dietary NO 3 − supplementation on NO 3 − metabolism, peripheral and central BP, pulse wave variables, and exercise performance, and the extent to which any improvements in these variables after NO 3 − supplementation exhibited a diurnal variation.It was hypothesised that NO 3 − supplementation would increase salivary and plasma [NO 2 − ], and improve brachial and central BP, pulse wave variables and exercise performance to a greater extent when ingested in the afternoon compared to the morning and evening.

Participants
Twelve young healthy males [mean ± SD: age: 23 ± 4 years, stature: 1.80 ± 0.09 m, body mass: 75.8 ± 10.9 kg, VO 2peak : 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.

Pre-visit standardisation
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 NO 3 −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 NO 3 − 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.

Experimental design
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 VO 2peak .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.VO 2peak 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 [NO 3 − ] and [NO 2 − ].Urine and serum osmolality were also evaluated at baseline to assess hydration status.Subsequently, participants ingested 2 × 70 mL of concentrated NO 3 − -rich (BR; 13 mmol NO 3 − ) or NO 3 − -depleted (PL; ~ 0.04 mmol NO 3 − ) 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 [NO 2 − ] ( 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, repeatedmeasures, 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.

Hydration status
Urine samples obtained at baseline was analyzed immediately to evaluate urine osmolality (Osmocheck, Vitech Scientific, UK), with a reading of < 700 mOsmol/kg required for visit continuation (Sawka et al. 2007).Additionally, 5 mL of venous blood was collected into a serum tube and left to clot at room temperature, with serum separated by centrifugation (3500 × g at 4 °C for 15 min) and frozen at − 80 °C for later analysis of osmolality via freezing-point depression (Gonotec 225 Osmomat 030 Cryoscopic Osmometer; Gonotec, Germany).Serum osmolality values ranging between 285 and 295 mOsmol .kg H 2 O −1 were taken to imply euhydration (Knepper et al. 2015).

Saliva collection
Prior to sample collection, participants rinsed their oral cavity with room-temperature tap water to remove any food debris.Following 2 min rest, unstimulated saliva samples were collected via passive drool and spit into pre-weighed sterile containers every 20 s for 2 min.This process was then repeated after 2 min.Samples were subsequently weighed for determination of SFR before salivary pH was measured in duplicate using a microFET electrode (Sentron, Leek, The Netherlands), with the measured pH value accepted once readings on the pH meter were stable for 5 s.A 3-point calibration of the pH probe was undertaken prior to analysis using buffers with known pH (4.01, 7.00, 10.01). 1 mL aliquots were then frozen at − 80 °C for later analysis of salivary [NO

Blood pressure
Participants were required to rest supine for 10 min.Thereafter, BP of the brachial artery was measured using an automated sphygmomanometer (Omron Healthcare, Kyoto, Japan).In total, five measurements were taken at 2 min intervals, with the mean of all five readings used for analysis.MAP was calculated as ([(2 × DBP) + SBP]/3).

Aortic blood pressure and pulse wave variables
Following 20 min supine rest, pulse wave analysis was assessed at the radial artery using applanation tonometry methods (SphygmoCor; Atcor Medical, Sydney, Australia) to determine central BP and indices of arterial stiffness.Pulse wave analysis calibrated to brachial BP uses a validated generalised transfer function to derive corresponding central aortic pressures (Chen et al. 1997).All tonometry data were recorded by a single investigator.A minimum of two recordings were taken at each time interval and the two measurements with the highest quality index (> 80%) were accepted for analysis.Pulse wave analysis indices of interest included: aortic systolic and diastolic BP, augmentation pressure (AP: the amplitude of the reflected wave), augmentation index (AI: the reflected wave amplitude divided by pulse pressure expressed as a percentage) and, due to the known influence of HR on AI, AI adjusted for heart rate of 75 bpm (AI@HR75).

Blood collection
Following 30 min supine rest, a tourniquet was applied around the upper arm prior to sample collection.Blood samples were subsequently drawn from an antecubital vein via venepuncture into 6 mL lithium heparin vacutainers.Samples were centrifuged at 3000 × g and 4 °C for 10 min, within 2 min of collection.Plasma was subsequently extracted and immediately frozen at − 80 °C for later analysis of [NO 3 − ] and [NO 2 − ].

Exercise procedures
All exercise tests were performed on an electronicallybraked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands).During the first laboratory visit, participants completed a ramp incremental test involving 4 min of baseline cycling at 20 W followed by a linear 30 W/min increase in work rate until task failure.Task failure was recorded once the pedal rate fell ≥ 10 rpm below self-selected cadence (70-100 rpm) for ≥ 5 s.The saddle and handlebar height and configuration were recorded and reproduced in subsequent tests.Breath-by-breath pulmonary gas exchange data were collected continuously during the incremental test and averaged over consecutive 10 s periods (Vyntus CPX metabolic cart, Vyaire Medical, Chicago, USA).Participants wore a face mask and breathed through a low dead space, low resistance, digital volume transducer assembly.The inspired and expired gas volume and gas concentration signals were continuously sampled via a capillary line connected to the mouthpiece.The gas analyser was calibrated prior to testing with gases of known concentration.
The turbine volume transducer was calibrated automatically and manually using a 3 L syringe (Hans Rudolph, Kansas City, Missouri).VO 2peak was taken as the highest 30 s mean value attained prior to the participant's volitional exhaustion.GET, was determined from a cluster of measurements including (1) the first disproportionate increase in CO 2 production ( VCO 2 ) from visual inspection of individual plots of VCO 2 vs. VO 2 , (2) an increase in expired ventilation ( VE )/ VO 2 with no increase in VE/ VCO 2 , and (3) an increase in end-tidal O 2 tension with no fall in end-tidal CO 2 tension.The TTE protocols involved 4 min cycling at 20 W followed by a step increase in work rate equivalent to 75%Δ (GET + 75% of the difference between the work rate at GET and PAP), with account taken for the mean response time for VO 2 during the ramp protocol (i.e., two-thirds of the ramp rate (20 W) deducted from the work rate at GET and PAP to account for the muscle-to-lung gas transit time).The test was terminated once pedal cadence fell ≥ 10 rpm below the self-selected cadence for ≥ 5 s.This exercise protocol was replicated during visits 3-8 and TTE was recorded.

[NO 3 − ] and [NO 2 − ] determination
All glassware, utensils and surfaces were rinsed thoroughly with deionised water to remove residual NO 3 − and NO 2 − prior to analysis.Plasma samples were deproteinised prior to [NO 3 − ] determination.Firstly, 500 μL of 0.18 N NaOH was added to 100 µL of sample followed by 5 min incubation at room temperature.Subsequently, samples were treated with 300 μL aqueous ZnSO4 (5% w/v) and vortexed for 30 s before undergoing an additional 10 min incubation period at room temperature.Samples were then centrifuged at 21,000 × g for 10 min and the supernatant was removed for subsequent analysis.The [NO 3 − ] of the deproteinised plasma sample was determined by its reduction to NO in the presence of 0.8% (w/v) vanadium chloride (VCl 3 ) in 1 M HCl via 50 μL injections into the septum of the air-tight purge vessel.The spectral emission of electronically excited nitrogen dioxide, derived from the reaction of NO with ozone, was detected by a thermoelectrically cooled, red-sensitive photomultiplier tube housed in a gas-phase chemiluminescence NO analyser (Sievers NOA 280i, Analytix Ltd, Durham, UK).All samples were analyzed in duplicate.The [NO 3 − ] was determined by plotting signal (mV) area against a calibration plot of sodium nitrate standards.Prior to plasma [NO 2 − ] determination, samples were deproteinised using ice-cold ethanol.Specifically, 500 μL of ethanol was added to 500 μL of sample followed by 15 min incubation.Samples were then centrifuged at 21,000 × g for 10 min and the supernatant was removed for subsequent analysis.Plasma [NO 2 − ] was determined by its reduction to NO in the presence of glacial acetic acid and aqueous sodium iodide (4% w/v) and calibrated using sodium nitrite standards.To determine plasma [NO 2 − ], 200 μL of deproteinised plasma was injected into the purge vessel.Origin Lab was used to smooth the NO analyser signal and objectively identify the peaks to derive the NO 2 − concentration data.After thawing at room temperature, saliva samples were centrifuged for 10 min at 21,000 × g and the supernatant was then removed and diluted at least 100fold with deionised water for subsequent analysis.[NO 3 − ] and [NO 2 − ] were determined using the same reagents described above for the respective plasma analyses.

Statistical analysis
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 repeatedmeasures ANOVAs.Plasma [NO 3 − ] and [NO 2 − ], 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 [NO 3 − ] and [NO 2 − ] 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 p 2 ) 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.

Hydration biomarkers
For all participants, urine osmolality on arrival was < 700 mOsmol .kg H 2 O −1 in all six conditions.Serum osmolality was between 285 and 295 mOsmol kg H 2 O −1 across all visits and not different between conditions (P > 0.050, n p 2 = 0.03).

Salivary flow rate and pH
There were no inter-condition differences in SFR or salivary pH at baseline (both P > 0.050).Mean SFR and pH between 1 and 2.5 h did not differ between conditions (n p 2 = 0.14, n p 2 = 0.16, both P > 0.050, Fig. 1, respectively).

Systolic blood pressure
There were no inter-condition differences in brachial SBP at baseline (P > 0.050).There was a main effect for time (P = 0.007, n p 2 = 0.50), but no main effect for condition (P > 0.050, n p 2 = 0.05) or condition × time interaction (P > 0.050, n p 2 = 0.09, Table 1).
Central diastolic blood pressure There were no inter-condition differences in central DBP at baseline (P > 0.050).There was no main effect for condition (P > 0.050, n p 2 = 0.02) or time (P > 0.050, n p 2 = 0.02), but there was a condition × time interaction effect (P = 0.011, n p 2 = 0.25).Post hoc analysis revealed no significant differences between conditions or over time (P > 0.050, Table 2).

Augmentation pressure, augmentation index and augmentation index normalised to heart rate
There were no inter-condition differences in AP, AI or AI@HR75 at baseline (all P > 0.050).There was no main effect for condition (P > 0.050, n p 2 = 0.06) or time (P > 0.050, n p 2 = 0.05) but there was a significant condition × time interaction (P = 0.029, n p 2 = 0.22) for AP.AP was lower at 2.5 h vs baseline in BR-AFT (P = 0.045, n p 2 = 0.89), but no differences were observed in the other conditions (all P > 0.050, Table 2).There was no main effect for condition (P > 0.050, n p 2 = 0.08) or time (P > 0.050, n p 2 = 0.17) for AI; however, there was a condition × time interaction (P = 0.022, n p 2 = 0.23).Post hoc analysis revealed no significant differences between conditions (P > 0.050, Table 2).There was a main effect for time (P = 0.024, n p 2 = 0.42) for AI@HR75, but no main effect for condition (P > 0.050, n p 2 = 0.04) or condition × time interaction (P > 0.050, n p 2 = 0.19, Table 2).

Discussion
This study assessed whether the time-of-day an acute dose of dietary NO 3 − was administered influenced its efficacy to lower BP and improve exercise performance in healthy adults.The principal novel findings from this study were: (1) SFR, salivary pH, BP and exercise performance did not exhibit a marked circadian rhythm; (2) salivary and plasma [NO 3 − ] and [NO 2 − ] were increased by a similar magnitude after BR ingestion in the morning, afternoon and evening; (3) BR consumption lowered central SBP by a similar magnitude across the day but did not reduce brachial SBP; and  (4) severe-intensity cycling TTE was not improved with BR supplementation irrespective of the time of day ingested.These findings improve understanding of the effects of acute BR supplementation on BP, vascular function and exercise performance in healthy young men by evaluating the potential for time-specific effects of BR supplementation on these health indices.

Salivary flow rate and pH
Previous studies have shown both unstimulated SFR (Dawes 1975(Dawes , 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(Dawes , 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.

Dietary nitrate metabolism
Salivary and plasma [NO 3 − ] and [NO 2 − ] 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 [NO 3 − ] and [NO 2 − ] increased following the acute ingestion of BR in the present study.However, contrary to the experimental hypothesis, the increases in salivary and plasma [NO 3 − ] and [NO 2 − ] 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 [NO 3 − ] (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 NO 3 − 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 [NO 3 − ] and [NO 2 − ] if assessed using more ecologically valid experimental designs (i.e., where individuals maintain their habitual dietary intake and activity levels) due to the NO 3 − content of the diet with different meals.

Blood pressure and vascular function
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 NO 3 − supplementation to lower BP, with a lowering in brachial SBP after NO 3 − 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 NO 3 − 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 NO 3 − 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 NO 3 − 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 [NO 2 − ] after BR ingestion were consistent across timepoints in the current study.Therefore, while previous studies have reported a strong agreement between plasma [NO 2 − ] and brachial SBP after acute NO 3 − supplementation, the current study suggests that central, but not brachial, SBP is modulated by circulating plasma [NO 2  − ].The mechanisms for the lowering of brachial SBP after NO 3 − ingestion have been considered to be linked to the reduction of circulating plasma NO 2 − to NO leading to elevated cyclic guanosine monophosphate signalling leading to vasodilation (Kapil et al. 2020(Kapil et al. , 2010)).However, NO 2 − can directly elicit vasodilation via s-nitrosylation (Bryan et al. 2005) 2015).It has also been suggested that the lowering in BP with elevated NO 2 − 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 NO 3 − 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 NO 3 − 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 NO 3 − 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 NO 3 − for 6 months (Faconti et al. 2019).Although exact mechanisms of action are unclear, lowered SBP yet unaltered arterial stiffness with NO 3 − consumption has been postulated to be due in part to increased venodilation leading to decreased preload (Mills et al. 2020).

Exercise performance
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(Chtourou et al. , 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 NO 3 − did not improve TTE during high-intensity cycling in the current study.While the existing literature generally supports a small but significant effect of NO 3 − supplementation to improve performance across a range of exercise settings (Senefeld et al. 2020), and acute NO 3 − 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 NO 3 − ) 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 NO 3 − dose (Wylie et al. 2013).There is some evidence to suggest that NO 3 − 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 [NO 2 − ] ( 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.

Potential implications and experimental considerations
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).et al. 2013).To accommodate for the slow plasma [NO 2 − ] pharmacokinetics after NO 3 − 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 midafternoon (~ 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.

Conclusion
In young healthy males, we observed no significant circadian variability in SFR, salivary pH, salivary and plasma ] and reductions in central SBP in the morning, afternoon, and evening.In contrast, brachial SBP was unchanged following BR supplementation in the morning, afternoon, and evening and TTE was not improved at any of the timepoints assessed after BR ingestion.These findings improve our understanding of the effect of BR supplementation on BP, vascular function and exercise performance and suggest that central SBP is consistently lowered across the day after BR supplementation in healthy adults.
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Fig. 1
Fig. 1 Mean salivary flow rate (SFR; upper panel) and salivary pH (lower panel) from 1 to 2.5 h following ingestion of nitrate-depleted and nitrate-rich beetroot juice in the morning (PL-MORN and BR-MORN), afternoon (PL-AFT and BR-AFT) and evening (PL-EVE and BR-EVE).The bars represent the group mean ± SEM responses with the filled circles representing individual participants.No differences observed between conditions (P > 0.050)

Fig
Fig. 2 Salivary nitrate concentration ([NO 3 − ], upper panel) and salivary nitrite concentration ([NO 2 − ], lower panel) at baseline, 1 h and 2.5 h following ingestion of nitrate-depleted and nitrate-rich beetroot juice in the morning (PL-MORN and BR-MORN), afternoon (PL-AFT and BR-AFT) and evening (PL-EVE and BR-EVE).Data presented as the group mean ± SEM responses with the filled circles representing individual participants.*Denotes higher than PL-MORN, PL-AFT and PL-EVE in BR-MORN, BR-AFT and BR-EVE (P < 0.050).#denotes salivary [NO 3 − ] lower than baseline at 2.5 h in PL-AFT and PL-EVE (P < 0.050)

Fig. 4
Fig. 4 Time to exhaustion (TTE) during severe-intensity cycling exercise following ingestion of nitrate-depleted or nitrate-rich beetroot juice in the morning (PL-MORN and BR-MORN), afternoon (PL-AFT and BR-AFT) and evening (PL-EVE and BR-EVE) (upper panel).The bars represent the group mean ± SEM responses with the filled circles representing individual participants.No differences observed between conditions (P > 0.050) post ingestion, and assessments of BP and exercise performance are recommended to take place upon attainment of peak plasma [NO 2 − ] (Wylie brachial or central BP or high-intensity exercise TTE across the non-supplemented baseline assessments.Acute NO 3 − -rich BR consumption resulted in similar increases in salivary and plasma [NO 3 − ] and [NO 2 −