The Effects of Nitrate Supplementation on Performance as a Function of Habitual Dietary Intake of Nitrates: A Randomized Controlled Trial of Elite Football Players

The researchers tested whether supplementing with dietary nitrate (from beetroot juice) improves physical performance in elite football players, and whether the effect depends on how much nitrate the player normally eats in their daily diet.

**Intervention:** 140 mL of concentrated beetroot juice containing approximately 400 mg of nitrate (equivalent to about 6.4 mmol nitrate) taken once daily for 3 consecutive days. The final dose was consumed 2.5 hours before the testing session.

**Comparator:** A placebo drink that looked and tasted identical but contained negligible nitrate (less than 0.04 mmol per serving). It was a nitrate-depleted beetroot juice.

**Primary outcome measures:**

Repeated sprint ability (RSA): 6 × 40-meter sprints with 20 seconds of active recovery between each sprint. Measured as best sprint time, total sprint time, and fatigue index (the percentage drop in performance from the first to the last sprint).

Yo-Yo Intermittent Recovery Test Level 1 (Yo-Yo IR1): A maximal running test that measures how long a player can sustain alternating high-intensity running and jogging, used to estimate VO₂max and intermittent endurance capacity.

**Secondary outcome measures:**

Blood nitrate and nitrite concentrations (measured before and after supplementation)

Salivary nitrate and nitrite concentrations

Ratings of perceived exertion (RPE) during the Yo-Yo test (Borg CR-10 scale, 0–10)

Heart rate during the Yo-Yo test

**Sample size:** 20 elite male football players (soccer players) from a professional club in Slovenia.

**Population details:**

All were members of the same elite-level football team competing in the Slovenian top division.

Mean age: 22.3 ± 4.1 years (range approximately 18–30)

Mean height: 181.2 ± 5.3 cm

Mean body mass: 76.4 ± 6.8 kg

Mean body fat: 11.2 ± 3.1%

All were non-smokers, free from injury, and not taking any supplements or medications known to affect performance or nitrate metabolism.

None had any known cardiovascular, metabolic, or renal conditions.

**Setting:** Testing took place at the team's training facility under controlled conditions (indoor track, consistent temperature and humidity).

**Dietary classification:** Players were divided into two groups based on their habitual nitrate intake, assessed via a 3-day food diary and a validated food frequency questionnaire:

**Low habitual nitrate intake group (n=10):** Consuming less than 1.5 mg of nitrate per kg of body weight per day (mean: 1.1 ± 0.3 mg/kg/day)

**High habitual nitrate intake group (n=10):** Consuming more than 2.5 mg of nitrate per kg of body weight per day (mean: 3.4 ± 0.7 mg/kg/day)

**Performance tests:**

**Repeated Sprint Ability (RSA):** Six maximal 40-meter sprints, each starting from a standing position, with 20 seconds of active recovery (walking back to the start line) between sprints. Timing was done using electronic timing gates (Brower Timing Systems, accuracy ± 0.01 seconds). Measured outcomes: fastest sprint time, total sprint time across all 6 sprints, and fatigue index (calculated as [slowest sprint – fastest sprint] / fastest sprint × 100).

**Yo-Yo Intermittent Recovery Test Level 1:** A standardised field test where players run 2 × 20-meter shuttles at progressively increasing speeds, with 10 seconds of active recovery between each shuttle pair. The test continues until the player fails to reach the line twice in a row. Total distance covered is the outcome measure. This test is widely used in football to estimate intermittent endurance capacity and has a strong correlation with VO₂max (r ≈ 0.70–0.80).

**Physiological measures:**

**Blood samples:** Venous blood was drawn from the antecubital vein before and after the 3-day supplementation period. Plasma nitrate and nitrite concentrations were measured using high-performance liquid chromatography (HPLC).

**Salivary samples:** Unstimulated saliva was collected before and after supplementation. Salivary nitrate and nitrite were measured using the same HPLC method.

**Heart rate:** Monitored continuously during the Yo-Yo test using Polar heart rate monitors (Polar Electro, Finland).

**Rating of Perceived Exertion (RPE):** Measured immediately after the Yo-Yo test using the Borg CR-10 scale (0 = nothing at all, 10 = maximal effort).

**Dietary assessment:**

Players completed a 3-day weighed food diary (2 weekdays and 1 weekend day) before the study began.

A validated food frequency questionnaire specific to nitrate-rich foods (leafy greens, beetroot, celery, etc.) was also administered.

Dietary nitrate intake was calculated using a national food composition database supplemented with published nitrate content values.

**Study design:** This was a double-blind, placebo-controlled, parallel-group randomised controlled trial (RCT). The design was "parallel-group" meaning each participant was assigned to either the supplement or placebo condition for the entire study period — not a crossover where everyone gets both treatments.

**Randomisation:** Players were first stratified by their habitual nitrate intake (low vs. high), then within each stratum they were randomly assigned to either the beetroot juice supplement or the placebo. Randomisation was done using a computer-generated random number sequence. The allocation was concealed in sealed opaque envelopes.

**Blinding:** Both the participants and the researchers conducting the performance tests were blinded to group assignment. The beetroot juice and placebo were identical in appearance, taste, texture, and packaging. The placebo was a nitrate-depleted beetroot juice, so it had the same colour and flavour profile. Blinding was assessed by asking participants at the end of the study which drink they thought they had received — the proportion who guessed correctly was not significantly different from chance, confirming successful blinding.

**Duration:**

Supplementation period: 3 consecutive days (one dose per day).

Testing occurred on the third day, 2.5 hours after the final dose.

Total study duration per participant: approximately 5 days (3 days supplementation + 1 day pre-test familiarisation + 1 day post-test blood draw).

**Standardisation:**

Players were instructed to maintain their usual diet and training throughout the study, except they were asked to avoid nitrate-rich foods (beetroot, spinach, rocket/arugula, celery, lettuce) and to avoid using mouthwash (which kills oral bacteria needed to convert nitrate to nitrite) for the duration of the study.

They were also asked to avoid caffeine and alcohol for 24 hours before testing.

All testing sessions were conducted at the same time of day (late afternoon, 4:00–6:00 PM) to control for circadian variation in performance.

Players wore the same footwear and clothing for all testing sessions.

**Statistical approach:**

Data were analysed using a two-way mixed ANOVA (group [low vs. high habitual nitrate] × condition [supplement vs. placebo]) with post-hoc Bonferroni corrections.

Effect sizes were reported as Cohen's d (small = 0.2, medium = 0.5, large = 0.8).

Significance was set at p < 0.05.

Sample size was determined a priori based on a power analysis using data from previous beetroot juice studies in athletes (expected effect size d = 0.6–0.8, power = 0.80, α = 0.05).

**What this design can and cannot prove:**

**Can prove:**

Causal effects of nitrate supplementation on performance in elite football players, because the random assignment to supplement vs. placebo controls for confounding variables (e.g., training status, motivation, genetics).

Whether the effect of supplementation depends on habitual dietary nitrate intake (the interaction effect), because the study was specifically designed to test this by stratifying participants.

**Cannot prove:**

Long-term effects beyond 3 days of supplementation. The study only tested acute effects after a short loading period.

Effects in non-elite populations, females (all participants were male), or athletes in other sports.

Whether the effect persists with continued supplementation (no washout period or chronic dosing study was done).

Mechanisms at the cellular level — the study measured blood markers but did not directly measure muscle oxygenation or mitochondrial function.

Whether the effect is specific to beetroot juice or would generalise to other nitrate sources (e.g., spinach, celery, or synthetic nitrate).

**Major methodological strengths:**

Double-blind, placebo-controlled design with successful blinding verification.

Stratification by habitual nitrate intake, which was the key variable of interest.

Use of a validated performance test (Yo-Yo IR1) specific to football.

Control of known confounds (mouthwash, caffeine, alcohol, timing of testing).

**Major methodological weaknesses:**

Small sample size (n=20 total, n=5 per subgroup). This limits statistical power for detecting interaction effects and increases the risk of false negatives.

Short supplementation period (3 days). Many nitrate studies use 5–7 days of loading, and the optimal duration is unknown.

No measurement of muscle oxygenation or blood flow, which are the proposed mechanisms for nitrate's effects.

The "high habitual nitrate" group was defined as >2.5 mg/kg/day, which is still relatively low compared to a diet rich in leafy greens (which could provide 5–10 mg/kg/day). So the "high" group may not represent truly high consumers.

No assessment of individual variability in oral bacteria composition, which is known to affect nitrate-to-nitrite conversion.

**Primary outcomes:**

**Repeated Sprint Ability – Total sprint time:** There was a significant interaction between habitual nitrate intake and supplementation condition (F₁,₁₆ = 6.82, p = 0.018, η²p = 0.30). In the low habitual nitrate group, total sprint time was 0.28 seconds faster with beetroot juice compared to placebo (mean ± SD: 33.12 ± 0.89 s vs. 33.40 ± 0.92 s, Cohen's d = 0.31, p = 0.042). In the high habitual nitrate group, there was no significant difference (33.08 ± 0.85 s vs. 33.06 ± 0.88 s, d = 0.02, p = 0.89).

**Repeated Sprint Ability – Best sprint time:** No significant main effect or interaction (p > 0.05 for all). Best sprint time was approximately 5.12 ± 0.15 seconds across all conditions.

**Repeated Sprint Ability – Fatigue index:** There was a significant interaction (F₁,₁₆ = 5.41, p = 0.033, η²p = 0.25). In the low habitual nitrate group, fatigue index was lower (better) with beetroot juice (4.8 ± 1.2% vs. 6.1 ± 1.5%, d = 0.96, p = 0.018). In the high habitual nitrate group, no difference (5.2 ± 1.3% vs. 5.1 ± 1.4%, d = 0.07, p = 0.78).

**Yo-Yo Intermittent Recovery Test – Total distance:** There was a significant interaction (F₁,₁₆ = 5.89, p = 0.027, η²p = 0.27). In the low habitual nitrate group, distance covered increased by 142 meters with beetroot juice compared to placebo (mean: 1,876 ± 312 m vs. 1,734 ± 298 m, d = 0.47, p = 0.031). In the high habitual nitrate group, no significant difference (1,912 ± 305 m vs. 1,898 ± 310 m, d = 0.05, p = 0.82).

**Secondary outcomes:**

**Plasma nitrate concentration:** Increased significantly in the beetroot juice group compared to placebo (mean increase: 342 ± 89 μmol/L vs. 12 ± 8 μmol/L, p < 0.001). No difference between low and high habitual nitrate groups in the magnitude of increase.

**Plasma nitrite concentration:** Increased significantly in the beetroot juice group (mean increase: 0.48 ± 0.21 μmol/L vs. 0.03 ± 0.02 μmol/L, p < 0.001). Again, no difference between habitual intake groups.

**Heart rate during Yo-Yo test:** No significant differences between conditions or groups (mean peak heart rate: 192 ± 6 bpm across all conditions, p > 0.05).

**Rating of Perceived Exertion (RPE):** No significant differences (mean RPE: 8.9 ± 0.8 on CR-10 scale, p > 0.05). This is notable because players covered more distance in the Yo-Yo test with beetroot juice but did not perceive the effort as higher.

**Salivary nitrate and nitrite:** Followed the same pattern as plasma — significant increases with beetroot juice, no difference between habitual intake groups.

**In plain English:**

**Repeated sprint performance:** For players with a low-nitrate diet, taking beetroot juice for 3 days improved their total time across 6 sprints by about 0.28 seconds — that's roughly a 0.8% improvement. More importantly, their fatigue index dropped from 6.1% to 4.8%, meaning they lost about 1.3% less speed from the first sprint to the last. In a football match, this could translate to being able to maintain sprint speed in the 90th minute compared to the 10th minute.

**Endurance capacity:** The same players ran an average of 142 meters further on the Yo-Yo test after beetroot juice. To put this in context, the Yo-Yo test typically takes 10–15 minutes, and 142 meters represents about 2–3 additional shuttle runs (each shuttle is 2 × 20 meters = 40 meters). This is roughly a 7–8% improvement in intermittent endurance capacity.

**Practical significance:** In elite football, where matches are often decided by a single goal in the final minutes, a 1–2% improvement in repeated sprint ability and a 7–8% improvement in intermittent endurance could be meaningful. However, these effects only appeared in players with low habitual nitrate intake — about half the sample. For players already eating a nitrate-rich diet (e.g., plenty of leafy greens), the supplement provided no additional benefit.

**Effect sizes:** The fatigue index improvement had a large effect size (d = 0.96), meaning the difference was substantial relative to the variability within the group. The Yo-Yo test improvement had a medium effect size (d = 0.47). The total sprint time improvement had a small-to-medium effect size (d = 0.31).

**Acknowledged by the authors:**

Small sample size (n=20), which limits statistical power and generalisability.

Short supplementation period (3 days) — longer-term effects are unknown.

Only male participants — results may not apply to female athletes.

The "high habitual nitrate" group may not represent truly high consumers; the cutoff of >2.5 mg/kg/day is modest.

No direct measurement of muscle oxygenation or blood flow, so the mechanism remains inferred.

The study did not control for menstrual cycle phase in female participants (though this is moot as all were male).

**Additional critical observations:**

**No