Nitrate is found in some root vegetables such as beets and in green leafy vegetables such as spinach and lettuce. The typical Western diet provides about 110 mg/day nitrate (1, 2). After ingestion, nitrate is metabolized to nitric oxide and other bioactive nitrogen oxides through a complex pathway. Nitrate is absorbed in the small intestine and peak plasma levels can be measured 30–60 min post-intake with an effective half-life of about six hours (3). Nitrate is cleared by the kidneys, but about 25% of the nitrate is taken up and secreted by the salivary glands and is subsequently reduced to nitrite by commensal bacteria in the mouth (4). The nitrite is then swallowed, absorbed through the intestinal tract, and further reduced to nitric oxide and other nitrite intermediates by enzymatic and nonenzymatic mechanisms in the blood and tissues. The use of mouthwashes, antacids, and chewing gum can interfere with the microbiome in the mouth and the conversion of nitrate to nitrite.

Dietary nitrate has been linked to several health-related benefits including blood pressure reduction, improved vascular function, and modulation of inflammatory processes and immune cell function (3). Nitric oxide acts as a signaling molecule during exercise and has physiological effects including vasodilation to increase blood flow regulation of muscle contraction and glucose uptake regulation of cellular respiration. The primary performance benefit of nitrate supplementation appears to be a reduction in the energy cost of exercise and a corresponding modest improvement in endurance capacity (5–7). Studies reporting performance benefits used acute nitrate doses of 5–6 mmol (or about 300 mg), or chronic doses at about half that amount (8). Chronic nitrate supplementation has not been consistently linked to improvements in aerobic capacity or performance (9). Concerns have been raised about high nitrate intake by athletes and the formation of carcinogenic N-nitroso compounds, a process that may be inhibited by vitamin C (10–13). An acceptable daily intake (ADI) level of 3.7 mg nitrates per kg body weight has been recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (14). This recommendation includes all nitrate sources including plant foods, drinking water, and cured and processed meats, although a different approach is recommended for nitrate from plant food sources (15). Co-supplementation of nitrate with caffeine, vitamin C, and other food components may enhance potential beneficial effects from moderate intake of nitrate and minimize risks from N-nitroso compounds (16).

Nitric oxide is considered a potent anti-inflammatory mediator and inhibitor of leukocyte recruitment, a key feature of inflammatory responses (3). However, most of the evidence comes from rodent-based studies. Human studies investigating the effects of nitrate or nitrate-rich beetroot juice on exercise-induced inflammation and muscle damage have produced mixed results (8, 17–19). Part of the problem is that only a few basic outcome measures related to inflammation (e.g., C-reactive protein, IL-6, IL-8) have been utilized in these studies. This study used a multi-omics approach centered on targeted oxylipins and untargeted proteomics analysis to investigate the influence of 2-weeks dietary nitrate ingestion from a beet-based mixed supplement on exercise-induced inflammation. Oxylipins are upstream regulators of inflammation, increase strongly after prolonged and vigorous exercise, and exert both pro- and anti-inflammatory effects depending on the fatty acid substrate and enzyme system (20–24). Untargeted proteomics studies indicate that long endurance cycling and running cause perturbations in numerous immune system and inflammatory proteins (25–27). Previous studies by our research group indicate that exercise-induced changes in inflammatory oxylipins and proteins can be moderated through nutrition-based interventions (21–24, 28, 29). We hypothesized that a multi-omics approach would reveal that 2-weeks ingestion of a beet-based supplement would mitigate exercise-induced inflammation.

Table 1 summarizes characteristics for the n = 20 study participants (n = 14 males, n = 6 females) who completed the study protocol. Age, percent body fat, aerobic capacity (VO2max), and training volume were similar between the male and female cyclists. The pattern of change over time did not differ between the male and female cyclists for a key outcome measurement (total plasma oxylipins, supplement x time x sex interaction effect, p-value =0.835). Thus, outcome measures for this randomized, crossover study are presented for all participants combined.

Performance data for each trial are summarized in Table 2. As designed, the two trials were similar in all cycling performance measures including total distance cycled, and percent of maximal heart rates, watts, and oxygen consumption rates.

Data for delayed onset of muscle soreness (DOMS), muscle damage biomarkers, the neutrophil/lymphocyte ratio, and serum cortisol are summarized in Table 3. Time effects for each of these variables were significant but the patterns of increase did not differ significantly between the BEET and PL trials (interaction effects, p-values >0.05).

BEET compared to placebo intake was associated with a significant increase in the sum of plasma nitrate and nitrite (NA + NI) (time and interaction effects, both p < 0.001) (Figure 2). The increase in NA + NI was significantly different between the BEET and PL trials after 2-weeks supplementation and throughout at least 3 h of recovery from the cycling bout. Trial differences for NA + NI at 24 h post-exercise tended to be different (p = 0.089).

Plasma arachidonic acid (ARA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) increased significantly post-exercise (time effects, p < 0.001) with no significant interaction effects (all p > 0.50) (Supplementary Data Sheet 1). A total of 67 oxylipins were detected in study samples. Of these, 41 oxylipins increased significantly post-exercise (Supplementary Data Sheet 1). These 41 oxylipins were summed for a composite variable and analysis showed significant post-exercise increases without trial differences between BEET and PL (interaction effect, p = 0.166) (Cohen’s d = 0.219, immediately post-exercise) (Table 4). Three other composite variables were calculated including seven oxylipins generated from arachidonic acid and cytochrome P-450 (ARA-CYP), two abundant oxylipins generated from linoleic acid and CYP (9,10-DiHOME, 12,13-DiHOME), and two abundant oxylipins generated from lipoxygenase (LOX) (9-HODE, 13-HODE) (Table 4). Significant time effects were shown for each of these composite oxylipin variables but without significant interaction effects (Table 4) (Cohen’s d = 0.102, 0.221, 0.283, respectively, immediately post-exercise). Two anti-inflammatory oxylipins were significantly higher in the BEET versus PL trials (Figures 3A,B). These included the EPA-derived oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE) (time effect, p < 0.001, interaction effect, p = 0.016) and the DHA-derived oxylipin 4-hydroxydocosahexaenoic acid (4-HDoHE) (time effect p < 0.001, interaction effect, p = 0.010).

A total of 616 plasma proteins were identified with no missing data for 458 proteins across all time points (Supplementary Data Sheet 2). Two-way ANOVA analysis of the normalized data showed that 45 proteins were lower and 21 proteins were higher in the BEET versus PL trials (all p < 0.05). The heatmap and line graphs of the clustered proteins are shown in Figure 4, and the identity of the clustered proteins is summarized in Table 5. Functional enrichment and protein–protein interaction networks are depicted in Figure 5 and supported a linkage of BEET supplementation with reduced complement activation and inflammatory responses, metabolic process and negative regulation of biological processes, and responses to stimulus, and an increased regulation of insulin-like growth factors (IGF) and uptake of IGF binding proteins, and symbiotic interactions.

This study employed a strong research design to investigate the effects of 2-weeks intake of a mixed beet-based supplement (212 mg nitrates per day) on inflammation induced by a 2.25 h cycling bout with 20 cyclists. A multi-omics approach was used, and this included a comprehensive targeted oxylipins panel and the measurement of several hundred proteins using untargeted proteomics. BEET supplementation significantly increased plasma NO₃⁻ + NO₂⁻ 1.6-fold after the 2-week supplementation period and 2.6-fold above placebo levels immediately after the cycling bout. The 2.25 h cycling bout caused significant inflammation with a composite variable of 41 oxylipins rising 3.4-fold immediately post-exercise above baseline plasma levels. Plasma oxylipins were still elevated 1.5-fold at the 3 h-post-exercise time point before falling to near baseline levels after 24 h recovery. BEET supplementation did not reduce post-exercise plasma concentrations of pro-inflammatory oxylipins but did have a modest effect in elevating two anti-inflammatory EPA- and DHA-derived oxylipins (18-HEPE and 4-HDoHE). Two-way ANOVA revealed 66 proteins that were increased or decreased with BEET supplementation, and functional enrichment supported significant BEET-related reductions in inflammation-related proteins including proteins related to complement activation, the acute phase response, and immune cell adhesion, migration, and differentiation.

This is the first exercise-based human clinical trial to investigate the influence of BEET supplementation on inflammation using a human systems biology approach. Other similar studies used a few targeted outcomes, had widely disparate research designs, and generally reported null or minor effects of increased plasma NO₃⁻ + NO₂⁻ from nitrate supplementation on post-exercise inflammation biomarkers including cytokines (8, 17–19). In one study with 34 runners, beetroot juice versus placebo supplementation for 3 days following a marathon race had no effect on muscle soreness, creatine kinase, interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNFα), blood leukocyte cell counts, or C-reactive protein (CRP) (19). The major limitation was that beetroot juice supplementation was restricted to the 3-day period after running the marathon race when most of the inflammation biomarkers were similar to pre-marathon levels.

Nearly all inflammation regulatory oxylipins generated in response to exercise stress come from PUFA substrates that are released from cell membranes and subsequently oxidized from CYP, LOX, and COX enzyme systems (20). Oxylipin production during recovery from prolonged and intensive cycling is sensitive to nutritional interventions including carbohydrate and blueberry supplementation (20–24). Underlying mechanisms are still being explored but may include the influence of glucose and polyphenol metabolites on CYP, LOX, and COX enzyme activity and the subsequent generation of specific oxylipin subgroups (21–24). Nitric oxide in cell culture and rodent-based studies inhibits P450 and LOX enzymes through multiple pathways, and can inhibit or stimulate COX enzymes depending on the physiological context (35, 36). Nitric oxide and oxylipins are viewed as interdependent signaling systems that help regulate inflammation, immune, and metabolic responses (37). Human clinical trials focused on nitrate supplementation and inflammation regulation from nitric oxide and oxylipins, however, are lacking.

In our study, BEET supplementation had a modest effect in increasing post-exercise EPA-COX derived HEPE-18 and DHA-LOX derived 4-HDoHE but had no significant effects on the generation of pro-inflammatory linoleic acid (LA)-CYP, LA-LOX, or arachidonic (ARA)-CYP oxylipins. These results suggest that pro-inflammatory oxylipin generation after 2.25 h intensive cycling is relatively unaffected by increases in plasma NO₃⁻ + NO₂⁻ with 2-weeks BEET intake. Whether or not higher and/or longer duration BEET or nitrate dosing may alter these results remains to be determined. In two previous studies, we showed that 14–18 days supplementation with 1 cup equivalent of blueberries had significant effects on post-exercise plasma oxylipin concentrations (21, 23). In one of these studies, blueberry ingestion was linked to a sustained elevation in DHA- and EPA-derived anti-inflammatory oxylipins including 18-HEPE in response to a 90-min bout of unaccustomed exercise by untrained adults (21). 18-HEPE is a precursor for resolvins that are specialized pro-resolving lipid mediators (SPM) with well-defined roles in inflammation resolution (38). 4-HDoHE is a beneficial 5-LOX oxidation product from DHA. Limited data indicate that this oxylipin in cell culture exerts anti-oxidative and anti-inflammatory effects on brain and blood vessel tissues through nuclear factor erythroid 2–related factor 2 (Nrf2) activation (39, 40).

Previous studies using untargeted proteomics methods indicate that acute prolonged and intensive exercise bouts have a widespread effect on plasma concentrations of proteins involved in immune system and inflammation responses (22, 25–27, 41, 42). The use of untargeted proteomics in studies with a sports nutrition focus is an emerging research design approach (22, 43). In a recent untargeted proteomics-based study, our research group showed that 4-weeks supplementation with the keto-carotenoid astaxanthin (8 mg/day) countered post-exercise decreases in humoral immunity and plasma immunoglobulins in athletes engaging in a vigorous 2.25 h running bout (22). In the current study, untargeted proteomics revealed an extensive effect of BEET versus placebo supplementation on 66 proteins, with lower plasma levels for a cluster of 45 proteins and higher levels for a cluster of 21 proteins. Functional enrichment supported a significant reduction in exercise-induced complement activation and inflammatory and stimulus responses. Key inflammation-related proteins that were lower with BEET supplementation included complements C3, C5, and C9, properdin (a positive regulator of the alternate pathway of complement), ficolin-3 (innate immune activator through the lectin complement pathway), serum amyloids A1 and P (acute phase proteins), alpha-1-microglobulin (an inflammation biomarker with acute phase proteins), clusterin (diverse inflammation roles), fibulin-1 and fermitin family homolog 3 (both involved with cell adhesion, migration, and differentiation), hypoxia up-regulated protein 1 (heat shock protein), coronin-1C (plays an important role in immune cell motility and vesicle trafficking), peroxiredoxin-1 (anti-oxidant that can promote inflammation), peroxisome proliferator-activated receptor-delta (regulates cellular metabolic functions and can promote inflammation), reelin (pro-inflammatory and pro-thrombotic factor), and dipeptidyl peptidase 4 membrane form (regulator of T-cell proliferation and natural killer cell kappa B activation). The liver is the source of most complement and acute phase proteins, and complement activation after prolonged and intensive exercise is an acute phase response to physiological stress (44). Nitric oxide is a pleiotropic and pervasive signaling molecule and influences inflammation in multiple ways including the suppression of immune cell growth, cytokine production, and platelet activation, and regulation of NF-κB activation (45).

Table 5 lists 14 immunoglobulin proteins that were lower with BEET versus placebo supplementation. Functional enrichment through STRING does not include immunoglobulins. Although human data are limited, emerging results from animal-based studies indicate an inverse relationship between nitric oxide and immunoglobulin production (46–48). Elevated nitric oxide may have multiple negative influences on B cell function. Nitric oxide generated from inflammatory monocytes, for example, can decrease B cell survival and hence antibody production (47). Vaccine adjuvants are being developed to counter the negative effect of nitric oxide on antibody responses (48). Our finding that BEET intake was associated with lower immunoglobulin proteins in cyclists during recovery from vigorous exercise was unexpected and needs to be confirmed in future studies and then evaluated for clinical significance.

BEET supplementation was linked to an increase in proteins involved with regulation of insulin like growth factors (IGFs) and symbiotic interactions. The IGF-axis mediates many of the physiological effects of growth hormone and is involved in the regulation of cell growth, proliferation, and survival (49). IGF binding protein 2 (IGFBP2) modifies IGF-1 functions and this protein was elevated with BEET supplementation. Nitric oxide mediates some of the effects of IGF-1 on vasodilation, increased blood flow, and lowered blood pressure (50). Other proteins with symbiotic interactions were elevated with BEET supplementation including neural cell adhesion molecule 1 (NCAM1 or CD56), heat shock cognate 71 kDa protein (HSPA8), and annexin A5. These proteins contribute to many biological processes including cell growth and differentiation, signaling, protein homeostasis, and apoptosis (51–53). Alpha-2-HS-glycoprotein or fetuin-A is a multifunctional protein, has a complex involvement with inflammation depending on the physiological context, and can function as an anti-inflammatory acute phase protein (54). Intercellular adhesion molecule 1 (ICAM-1) is an adhesion receptor and was also elevated with BEET supplementation. ICAM-1 regulates inflammatory responses by controlling leukocyte recruitment from the blood compartment to sites of inflammation and also is involved in epithelial injury-resolution responses (55).

The strength of this study was the combination of a strong research design and multi-omics outcomes to determine if exercise-induced inflammation could be moderated with BEET supplementation. There are several research design features and limitations that should be considered when interpreting study results. This study included non-elite male and female cyclists who were generally middle-aged and cycled for 2 h 15 min at about 75% VO_2max. Thus, the results of this study may not apply to younger elite endurance athletes who exercise at higher intensities. The research design included a 2-week supplementation period of BEET or placebo (two doses per day) with intake continued the day of the exercise challenge. Thus, the changes in protein biomarkers reported in this study could be related to both acute and chronic BEET intake. This research design element is consistent with the practice of many athletes who consume supplements both during training and on the day of competition. Plant-based supplements undergo a complex metabolic process involving the oral and gut microbiomes, metabolite formation from substrates, and subsequent biochemical effects on various enzyme systems including those related to inflammation. Our blood samples were collected in an overnight fasted state (pre- and post-supplementation). On the exercise challenge day, the blood collection was followed by the normal BEET dose and then the cycling bout. Thus, subjects had not consumed the BEET or placebo supplement since the prior day (midday) and most likely explains the low pre-exercise plasma NO₃⁻ + NO₂⁻ levels. The primary purpose of this study was to investigate the effects of BEET supplementation on exercise-induced inflammation. For this reason, participants exercised in an overnight fasted state to avoid the interactive effects of carbohydrate intake on inflammation. The BEET supplement included a moderate amount of nitrate from beets and fermented beets with caffeine, vitamin C, and other food components that may minimize risks from N-nitroso compounds. Beetroot powder also contains other phytochemicals that may have bioactive effects. We cannot rule out the potential synergistic effects of these other ingredients with beetroot on inflammation.

This randomized crossover study used a 2.25 h cycling bout to induce inflammation. Using double blind, placebo-controlled methods, 2-weeks intake of a BEET-based supplement with a moderate amount of nitrate (212 mg/d) increased plasma levels of NO₃⁻ + NO₂⁻. BEET supplementation did not counter exercise-induced increases in pro-inflammatory oxylipins but did increase post-exercise levels of two anti-inflammatory EPA- and DHA-derived oxylipins. The strongest effect of BEET supplementation was related to a significant decrease in a cluster of proteins involved in complement activation and inflammation. These data support that 2-weeks intake of a mixed beet-based supplement with morning and midday doses moderates protein biomarkers of exercise-induced inflammation in athletes. Carbohydrate and blueberry ingestion also mitigate post-exercise inflammation, an effect that requires more investigation for biological and clinical significance, but is generally interpreted as beneficial to the athlete over the long term (21, 23, 24, 29). This study did not focus on testing different formulations or dosing approaches. Future research can establish whether higher beet-nitrate intake with various types of adjuvants over a longer time period can amplify the anti-inflammatory effects, especially on pro-inflammatory oxylipins.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The studies involving humans were approved by Appalachian State University Institutional Review Board, NIH. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

DN: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. CS: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. JW: Formal analysis, Investigation, Methodology, Writing – review & editing. FM: Data curation, Formal analysis, Methodology, Writing – review & editing. PS: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. AO: Data curation, Formal analysis, Investigation, Writing – review & editing. QZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Writing – review & editing.