The effects of nitrate on the oral microbiome: a systematic review investigating prebiotic potential.

Background: Nitrate (NO3-) has been suggested as a prebiotic for oral health. Evidence indicates dietary nitrate and nitrate supplements can increase the proportion of bacterial genera associated with positive oral health whilst reducing bacteria implicated in oral disease(s). In contrast, chlorhexidine-containing mouthwashes, which are commonly used to treat oral infections, promote dysbiosis of the natural microflora and may induce antimicrobial resistance. Methods: A systematic review of the literature was undertaken, surrounding the effects of nitrate on the oral microbiota. Results: Overall, n = 12 in vivo and in vitro studies found acute and chronic nitrate exposure increased (representatives of) health-associated Neisseria and Rothia (67% and 58% of studies, respectively) whilst reducing periodontal disease-associated Prevotella (33%). Additionally, caries-associated Veillonella and Streptococcus decreased (25% for both genera). Nitrate also altered oral microbiome metabolism, causing an increase in pH levels (n = 5), which is beneficial to limit caries development. Secondary findings highlighted the benefits of nitrate for systemic health (n = 5). Conclusions: More clinical trials are required to confirm the impact of nitrate on oral communities. However, these findings support the hypothesis that nitrate could be used as an oral health prebiotic. Future studies should investigate whether chlorhexidine-containing mouthwashes could be replaced or complemented by a nitrate-rich diet or nitrate supplementation.

Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability.

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P < 0.05 in all five datasets with n = 20-82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P < 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P < 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies.

Reduced nitric oxide synthesis in winter: A potential contributing factor to increased cardiovascular risk.

BACKGROUND: Nitric oxide is a key signalling molecule that elicits a range of biological functions to maintain vascular homeostasis. A reduced availability of nitric oxide is implicated in the progression of cardiovascular diseases and increases the risk of pathogenic events. AIMS: To compare the concentration of nitric oxide metabolites in healthy adults between winter and summer months. DESIGN: An observational study of healthy adults (age 32 ± 9 years) living in central Scotland. METHODS: Thirty-four healthy adults (13 females) were monitored for 7 days in summer and winter to record sunlight exposure (ultraviolet-A (UV-A) radiation), diet, and physical activity. At the end of each phase, blood pressure was measured, and samples of blood and saliva collected. The samples were analysed to determine the concentrations of plasma and salivary nitrate and nitrite and serum 25-hydroxyvitamin D (25(OH)D). RESULTS: The participants maintained similar diets in each measurement phase but were exposed to more UV-A radiation (550%) and undertook more moderate-vigorous physical activity (23%) in the summer than in winter. Plasma nitrite (46%) and serum 25(OH)D (59%) were higher and blood pressure was lower in the summer compared to winter months. Plasma nitrite concentration was negatively associated with systolic, diastolic, and mean arterial blood pressure. CONCLUSIONS: Plasma nitrite, an established marker of nitric oxide synthesis, is higher in healthy adults during the summer than in winter. This may be mediated by a greater exposure to UV-A which stimulates the release of nitric oxide metabolites from skin stores. While it is possible that seasonal variation in nitric oxide availability may contribute to an increased blood pressure in the winter months, the overall impact on cardiovascular health remains to be determined.

The oral microbiome, nitric oxide and exercise performance.

The human microbiome comprises ∼1013-1014 microbial cells which form a symbiotic relationship with the host and play a critical role in the regulation of human metabolism. In the oral cavity, several species of bacteria are capable of reducing nitrate to nitrite; a key precursor of the signaling molecule nitric oxide. Nitric oxide has myriad physiological functions, which include the maintenance of cardiovascular homeostasis and the regulation of acute and chronic responses to exercise. This article provides a brief narrative review of the research that has explored how diversity and plasticity of the oral microbiome influences nitric oxide bioavailability and related physiological outcomes. There is unequivocal evidence that dysbiosis (e.g. through disease) or disruption (e.g. by use of antiseptic mouthwash or antibiotics) of the oral microbiota will suppress nitric oxide production via the nitrate-nitrite-nitric oxide pathway and negatively impact blood pressure. Conversely, there is preliminary evidence to suggest that proliferation of nitrate-reducing bacteria via the diet or targeted probiotics can augment nitric oxide production and improve markers of oral health. Despite this, it is yet to be established whether purposefully altering the oral microbiome can have a meaningful impact on exercise performance. Future research should determine whether alterations to the composition and metabolic activity of bacteria in the mouth influence the acute responses to exercise and the physiological adaptations to exercise training.

Nitrate-rich beetroot juice offsets salivary acidity following carbohydrate ingestion before and after endurance exercise in healthy male runners.

There have been recent calls for strategies to improve oral health in athletes. High carbohydrate diets, exercise induced dehydration and transient perturbations to immune function combine to increase oral disease risk in this group. We tested whether a single dose of nitrate (NO3-) would offset the reduction in salivary pH following carbohydrate ingestion before and after an exercise bout designed to cause mild dehydration. Eleven trained male runners ([Formula: see text] 53 ± 9 ml∙kg-1∙min-1, age 30 ± 7 years) completed a randomised placebo-controlled study comprising four experimental trials. Participants ingested the following fluids one hour before each trial: (a) 140 ml of water (negative-control), (b) 140 ml of water (positive-control), (c) 140 ml of NO3- rich beetroot juice (~12.4 mmol NO3-) (NO3- trial) or (d) 140 ml NO3- depleted beetroot juice (placebo-trial). During the negative-control trial, participants ingested 795 ml of water in three equal aliquots: before, during, and after 90 min of submaximal running. In the other trials they received 795 ml of carbohydrate supplements in the same fashion. Venous blood was collected before and after the exercise bout and saliva was sampled before and repeatedly over the 20 min following carbohydrate or water ingestion. As expected, nitrite (NO2-) and NO3- were higher in plasma and saliva during the NO3- trial than all other trials (all P<0.001). Compared to the negative-control, salivary-pH was significantly reduced following the ingestion of carbohydrate in the positive-control and placebo trials (both P <0.05). Salivary-pH was similar between the negative-control and NO3- trials before and after exercise despite ingestion of carbohydrate in the NO3- trial (both P≥0.221). Ingesting NO3- attenuates the expected reduction in salivary-pH following carbohydrate supplements and exercise-induced dehydration. NO3- should be considered by athletes as a novel nutritional strategy to reduce the risk of developing acidity related oral health conditions.

Venous occlusion during blood collection decreases plasma nitrite but not nitrate concentration in humans.

BACKGROUND: To maintain vascular tone and blood flow when tissue oxygenation is reduced, nitrite anions are reduced to nitric oxide (NO). From a practical perspective, it is unclear how the application of a tourniquet during blood collection might influence measurement of NO metabolites. Accordingly, this study evaluated the effect of venous occlusion on plasma nitrite and nitrate during venous blood collection. METHODS: Fifteen healthy participants completed two trials that were preceded by the ingestion of nitrate-rich beetroot juice (BRJ; total of ~8.4 mmol nitrate) or no supplementation (control). In both trials, blood was collected using a venepuncture needle while a tourniquet was applied to the upper arm and using an indwelling intravenous cannula, from the opposing arm. The venepuncture samples were collected at 35 s post occlusion. Changes in the oxygenation of forearm flexor muscles were assessed using near-infrared spectroscopy. Plasma nitrite and nitrate were analysed using gas-phase chemiluminescence. RESULTS: In the control trial, plasma nitrite was significantly elevated when collected via the cannula (179 ± 67 nM) compared to venepuncture (112 ± 51 nM, P = 0.03). The ingestion of BRJ increased plasma nitrite and values remained higher when sampled from the cannula (473 ± 164 nM) compared to venepuncture (387 ± 136 nM, P < 0.001). Plasma nitrate did not differ between collection methods in either trial (all P > 0.05). The delta changes in total-, deoxy-, and oxy-haemoglobin were all significantly greater during venepuncture sample compared to the cannula sample at the point of blood collection (all P < 0.05). CONCLUSIONS: Venous occlusion during venepuncture blood collection lowers plasma nitrite concentration, potentially due to localised changes in haemoglobin concentration and/or a suppression of endogenous NO synthesis. Accordingly, the method of blood collection to enable measurements of NO metabolites should be carefully considered and consistently reported by researchers.

Effects of Chlorhexidine mouthwash on the oral microbiome.

Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.

Dietary nitrate supplementation alters the oral microbiome but does not improve the vascular responses to an acute nitrate dose.

Nitrate (NO3-) contained in food and beverages can transiently increase nitric oxide (NO) availability following a stepwise reduction to nitrite (NO2-) by commensal bacteria in the oral cavity. We tested the hypothesis that regular ingestion of dietary NO3- would influence the oral microbiome, the capacity to reduce NO3- to NO2- in saliva, and the vascular responses to an acute dose of NO3-. The abundance of bacterial species on the tongue, the availability of NO markers, and vascular function were assessed in 11 healthy males before and after 7 days of supplementation with NO3--rich beetroot juice and a NO3--depleted placebo. As expected, saliva and plasma NO2- and NO3- were significantly elevated after NO3- supplementation (all P < 0.05) but not placebo. We found that NO3- supplementation increased salivary pH (7.13 ±â€¯0.54 to 7.39 ±â€¯0.68, P = 0.043) and altered the abundance of some bacteria previously implicated in NO3- reduction: Neisseria (from 2% ±â€¯3%-9% ±â€¯5%, P < 0.001), Prevotella (from 34% ±â€¯17%-23% ±â€¯11%, P = 0.001) and Actinomyces (from 1% ±â€¯1%-0.5% ±â€¯0.4%). Despite these alterations to the oral microbiota, an acute dose of NO3- increased salivary and plasma NO2-, reduced systolic blood pressure and increased the response to flow mediated dilation to a similar extent before and after 7 days of supplementation (P > 0.05). Our study establishes that supplementing the diet with NO3- for a sustained period can alter the oral environment in favour of health but does not impact the response to an acute NO3- dose. Acute ingestion of NO3- results in transient improvements in vascular function but the dietary induced adaptations to the oral bacteria did not enhance these effects.

Dietary intake of inorganic nitrate in vegetarians and omnivores and its impact on blood pressure, resting metabolic rate and the oral microbiome.

Vegetarian diets are commonly associated with lower blood pressure levels. This has been related to greater consumption of inorganic nitrate, since vegetables are the main source of this anion. Dietary nitrate is reduced to nitrite by commensal bacteria in the mouth, which in turn leads to increased circulatory nitrite availability. Nitrite can form nitric oxide by several pathways promoting a reduction in the vascular tone and lower blood pressure. This study tested whether vegetarians have higher concentrations of nitrite in saliva and plasma, and lower blood pressure and resting metabolic rate (RMR), due to higher intakes of nitrate, compared to omnivores. Following a non-randomized, cross-over and single-blinded design we measured dietary nitrate intake, blood pressure and RMR in young and healthy vegetarians (n = 22) and omnivores (n = 19) with similar characteristics after using placebo or antibacterial mouthwash for a week to inhibit oral bacteria. Additionally, we analyzed salivary and plasma nitrate and nitrite concentrations, as well as the oral nitrate-reduction rate and oral microbiome in both groups. Dietary nitrate intake in vegetarians (97 ±â€¯79 mg/day) was not statistically different (P > 0.05) to omnivores (78 ±â€¯47 mg/day). Salivary and plasma nitrate and nitrite concentrations were similar after placebo mouthwash in both groups (P > 0.05). The oral nitrate-reducing capacity, abundance of oral bacterial species, blood pressure and RMR were also similar between vegetarians and omnivores (P > 0.05). Antibacterial mouthwash significantly decreased abundance of oral nitrate-reducing bacterial species in vegetarians (_16.9%; P < 0.001) and omnivores (_17.4%; P < 0.001), which in turn led to a significant reduction of the oral nitrate-reducing capacity in vegetarians (-78%; P < 0.001) and omnivores (-85%; P < 0.001). However, this did not lead to a significant increase in blood pressure and RMR in either groups (P > 0.05). These findings suggest that vegetarian diets may not alter nitrate and nitrite homeostasis, or the oral microbiome, compared to an omnivore diet. Additionally, inhibition of oral nitrite synthesis for a week with antibacterial mouthwash did not cause a significant raise in blood pressure and RMR in healthy, young individuals independent of diet.

Lower limb ischemic preconditioning combined with dietary nitrate supplementation does not influence time-trial performance in well-trained cyclists.

OBJECTIVES: Dietary nitrate (NO3-) supplementation and ischaemic preconditioning (IPC) can independently improve exercise performance. The purpose of this study was to explore whether NO3- supplementation, ingested prior to an IPC protocol, could synergistically enhance parameters of exercise. DESIGN: Double-blind randomized crossover trial. METHODS: Ten competitive male cyclists (age 34±6years, body mass 78.9±4.9kg, V⋅O2peak 55±4 mLkgmin-1) completed an incremental exercise test followed by three cycling trials comprising a square-wave submaximal component and a 16.1km time-trial. Oxygen uptake (V⋅O2) and muscle oxygenation kinetics were measured throughout. The baseline (BASE) trial was conducted without any dietary intervention or IPC. In the remaining two trials, participants received 3×5min bouts of lower limb bilateral IPC prior to exercise. Participants ingested NO3--rich gel (NIT+IPC) 90min prior to testing in one trial and a low NO3- placebo in the other (PLA+IPC). Plasma NO3- and nitrite (NO2-) were measured immediately before and after application of IPC. RESULTS: Plasma [NO3-] and [NO2-] were higher before and after IPC in NIT+IPC compared to BASE (P<0.001) but did not differ between BASE and PLA+IPC. There were no differences in V⋅O2 kinetics or muscle oxygenation parameters between trials (all P>0.4). Performance in the time-trial was similar between trials (BASE 1343±72s, PLA+IPC 1350±75s, NIT+IPC 1346±83s, P=0.98). CONCLUSIONS: Pre-exercise IPC did not improve sub-maximal exercise or performance measures, either alone or in combination with dietary NO3- supplementation.

Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference.

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CVB), and analytical imprecision (CVA) in experimental endpoints. However, no study has quantified the CVA and CVB of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CVB, CVA, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CVB of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CVB in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ±â€¯5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (∼12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitrate-reducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CVB for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CVB for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CVB for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CVB in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

Salivary nitrite production is elevated in individuals with a higher abundance of oral nitrate-reducing bacteria.

Nitric oxide (NO) can be generated endogenously via NO synthases or via the diet following the action of symbiotic nitrate-reducing bacteria in the oral cavity. Given the important role of NO in smooth muscle control there is an intriguing suggestion that cardiovascular homeostasis may be intertwined with the presence of these bacteria. Here, we measured the abundance of nitrate-reducing bacteria in the oral cavity of 25 healthy humans using 16S rRNA sequencing and observed, for 3.5 h, the physiological responses to dietary nitrate ingestion via measurement of blood pressure, and salivary and plasma NO metabolites. We identified 7 species of bacteria previously known to contribute to nitrate-reduction, the most prevalent of which were Prevotella melaninogenica and Veillonella dispar. Following dietary nitrate supplementation, blood pressure was reduced and salivary and plasma nitrate and nitrite increased substantially. We found that the abundance of nitrate-reducing bacteria was associated with the generation of salivary nitrite but not with any other measured variable. To examine the impact of bacterial abundance on pharmacokinetics we also categorised our participants into two groups; those with a higher abundance of nitrate reducing bacteria (> 50%), and those with a lower abundance (< 50%). Salivary nitrite production was lower in participants with lower abundance of bacteria and these individuals also exhibited slower salivary nitrite pharmacokinetics. We therefore show that the rate of nitrate to nitrite reduction in the oral cavity is associated with the abundance of nitrate-reducing bacteria. Nevertheless, higher abundance of these bacteria did not result in an exaggerated plasma nitrite response, the best known marker of NO bioavailability. These data from healthy young adults suggest that the abundance of oral nitrate-reducing bacteria does not influence the generation of NO through the diet, at least when the host has a functional minimum threshold of these microorganisms.

The effects of two different doses of ultraviolet-A light exposure on nitric oxide metabolites and cardiorespiratory outcomes.

PURPOSE: The present study investigated different doses of ultraviolet-A (UV-A) light on plasma nitric oxide metabolites and cardiorespiratory variables. METHODS: Ten healthy male participants completed three experimental conditions, 7 days apart. Participants were exposed to no light (CON); 10 J cm2 (15 min) of UV-A light (UVA10) and 20 J cm2 (30 min) of UV-A light (UVA20) in a randomized order. Plasma nitrite [NO2-] and nitrate [NO3-] concentrations, blood pressure (BP), and heart rate (HR) were recorded before, immediately after exposure and 30 min post-exposure. Whole body oxygen utilization ([Formula: see text]), resting metabolic rate (RMR) and skin temperature were recorded continuously. RESULTS: None of the measured parameters changed significantly during CON (all P > 0.05). [Formula: see text] and RMR were significantly reduced immediately after UVA10 (P < 0.05) despite no change in plasma [NO2-] (P > 0.05). Immediately after exposure to UVA20, plasma [NO2-] was higher (P = 0.014) and [Formula: see text] and RMR tended to be lower compared to baseline (P = 0.06). There were no differences in [NO2-] or [Formula: see text] at the 30 min time point in any condition. UV-A exposure did not alter systolic BP, diastolic BP or MAP (all P > 0.05). UV-A light did not alter plasma [NO3-] at any time point (all P > 0.05). CONCLUSIONS: This study demonstrates that a UV-A dose of 20 J cm2 is necessary to increase plasma [NO2-] although a smaller dose is capable of reducing [Formula: see text] and RMR at rest. Exposure to UV-A did not significantly reduce BP in this cohort of healthy adults. These data suggest that exposure to sunlight has a meaningful acute impact on metabolic function.

Changes in body posture alter plasma nitrite but not nitrate concentration in humans.

PURPOSE: This study evaluated the change (Δ) in plasma volume (PV), nitrate [NO3-], and nitrite [NO2-] concentration following changes in posture in the presence and absence of elevated plasma [NO3-] and [NO2-] METHODS: Fourteen healthy participants completed two trials that were preceded by either supplementation with NO3--rich beetroot juice (BR; total of ∼31 mmol NO3-) or no supplementation (CON). Both trials comprised 30 min of lying supine followed by 2 min of standing, 2 min of sitting and 5 min of sub-maximal cycling. Measurements of plasma [NO3-] and [NO2-] were made by gas-phase chemiluminescence and ΔPV was estimated using the Dill and Costill method. RESULTS: Plasma [NO2-] decreased from baseline (CON: 120 ± 49 nM, BR: 357 ± 129 nM) after lying supine for 30 min (CON 77 ± 30 nM; BR 231 ± 92 nM, both P < 0.01) before increasing during standing (CON 109 ± 42 nM; BR 297 ± 105 nM, both P < 0.01) and sitting (CON 131 ± 43 nM; BR 385 ± 125 nM, both P < 0.01). Plasma [NO2-] remained elevated following exercise only in CON (125 ± 61 nM P = 0.02). Plasma [NO3-] was not different between measurement points in either condition (P > 0.05). PV increased from baseline during the supine phase before decreasing upon standing, sitting, and exercise in both trials (all P<0.05). CONCLUSIONS: Changing body posture causes rapid and consistent alterations in plasma [NO2-]. Researchers should therefore carefully consider the effect of posture when measuring this variable.

Metabolomic Profiling of Submaximal Exercise at a Standardised Relative Intensity in Healthy Adults.

Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capacity (VO2max) was determined and the second was a 45 min submaximal exercise test. Urine samples were collected separately the day before (day 1) , the day of (day 2), and the day after (day 3) the submaximal exercise test (12 samples per subject). Metabolomic profiling of the samples was carried out using hydrophilic interaction chromatography (HILIC) coupled to an Orbitrap Exactive mass spectrometer. Data were extracted, database searched and then subjected to principle components (PCA) and orthogonal partial least squares (OPLSDA) modelling. The best results were obtained from pre-treating the data by normalising the metabolites to their mean output on days 1 and 2 of the trial. This allowed PCA to separate the day 2 first void samples (D2S1) from the day 2 post-exercise samples (D2S3) PCA also separated the equivalent samples obtained on day 1 (D1S1 and D1S3). OPLSDA modelling separated both the D2S1 and D2S3 samples and D1S1 and D1S3 samples. The metabolites affected by the exercise samples included a range of purine metabolites and several acyl carnitines. Some metabolites were subject to diurnal variation these included bile acids and several amino acids, the variation of these metabolites was similar on day 1 and day 2 despite the exercise intervention on day 2. Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO2max when the levels in the D2S3 samples were considered.