The Effects of Acute Nitrate and Caffeine Co-ingestion on Discipline-Specific Exercise Performance.

The Acute Effects of Dietary Nitrate (Beetroot Juice), Caffeine, and Their Combination on Discipline-Specific Exercise Performance, Lactate Kinetics, and Physiological Responses in Trained Functional Fitness Athletes and Elite Junior Swimmers.

The purpose of this study is to investigate the acute, separate, and combined ergogenic effects of dietary nitrate (beetroot juice, BJ) and caffeine (CAF) supplementation on discipline-specific athletic performance. The project employs a double-blind, randomized, placebo-controlled, four-arm crossover design. It evaluates two highly distinct, heterogeneous populations of athletes: (A) adult high-intensity functional training (HIFT/CrossFit/Hyrox) competitors performing the standardized Hyrox Physical Fitness Test (P'F'T), and (B) elite junior competitive swimmers (National Team members) performing a progressive 8 × 100 m swimming test. The study will assess physical performance, lactate kinetics, subjective exertion, biochemical indices, psychological metrics, and gastrointestinal tolerance.

Study Overview

• Status: Not yet recruiting
• Conditions: Athletic Performance; Dietary Supplement; Iron Status
• Intervention / Treatment: Dietary supplement: Caffeine; Dietary supplement: Dietary Nitrate (Beetroot Juice); Dietary supplement: Placebo (Nitrate-Depleted Beetroot Juice); Dietary supplement: Placebo (Maltodextrin Capsule)

Detailed Description

This project addresses a critical gap in sports nutrition literature regarding the potential synergistic, additive, or antagonistic interactions of caffeine (CAF) and dietary nitrate (beetroot juice, BJ) when co-administered. While CAF acts primarily as an adenosine receptor antagonist in the central nervous system to enhance motor unit recruitment and decrease perceived exertion, BJ acts as a physiological precursor to nitric oxide, improving peripheral muscle contractility, mitochondrial efficiency, and muscle perfusion.

To evaluate whether these effects are universal or discipline-specific, the trial employs a two-population cohort design:

• Cohort A (HIFT/Hyrox): Investigates adult functional fitness athletes (aged 18-45) characterized by concurrent strength and endurance adaptations. They will perform the ecological Hyrox P'F'T.
• Cohort B (Elite Junior Swimmers): Investigates elite junior national team swimmers (aged 16-19) during their centralized training camp. They will undergo the standardized progressive 8 × 100 m step-test.

Furthermore, this study will analyze whether individual hematological markers (iron status) and biochemical profiles correlate with the magnitude of the ergogenic response to establish prospective predictive biomarkers for customized sports nutrition.

• Study Type: Interventional
• Enrollment (Estimated): 112
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Natalia Główka, PhD; Phone Number: +48691756944; Email: glowka@awf.poznan.pl

Study Locations

• Poland
  • Wielkopolska
    • Poznan, Wielkopolska, Poland, 61-871
      • Poznan University of Physical Education
      • Contact: Natalia Główka; Phone Number: 691756944; Email: glowka@awf.poznan.pl

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

A) Cohort A (HIFT / CrossFit / Hyrox):

• Aged 18-45 years.
• Minimum of 2 years of systematic HIFT/CrossFit/Hyrox training experience.
• Performing >4 training sessions per week.
• Ability to perform the HYROX Physical Fitness Test (P'F'T) and has participated in at least one official HYROX event.
• Possession of a valid medical certificate proving eligibility for competitive sports.

B) Cohort B (Competitive Junior Swimmers):

• Aged 16-19 years.
• Active member of the Polish Junior National Swimming Team.
• Minimum of 5 years of competitive swimming experience.
• Current training volume of at least 8-10 pool training sessions per week.
• Written informed consent obtained from both the participant and their legal guardian (for minors under 18).

C)Common Criteria for Both Cohorts:

• No history of cardiovascular, respiratory, metabolic, or musculoskeletal disease contradicting high-intensity exercise or stimulant intake.
• No known food allergies or adverse reactions to beetroot products or caffeine.
• No current use of medications that alter nitrate/caffeine metabolism (e.g., proton pump inhibitors, antibiotics, phosphodiesterase inhibitors).
• History of uneventful habituation to caffeine use during training/competition.
• Willingness to abstain from dietary supplements, antibacterial mouthwashes, alcohol, and high-nitrate foods for 48 hours prior to each testing session.
• Good general health and physical well-being.

Exclusion Criteria (Applicable to both cohorts):

• Occurrence of acute musculoskeletal injury or trauma during the study period.
• Any medical or health contraindications (including severe menstrual disorders, planned pregnancy, or breastfeeding).
• Failure or refusal to strictly adhere to the dietary, supplementation, and exercise testing protocols.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Crossover Assignment
• Masking: Quadruple

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: CAF + BJ (Active Co-ingestion); Participants ingest a concentrated beetroot juice shot (~6.4 mmol / 400 mg nitrates) 3 hours before exercise, and a capsule of caffeine (Cohort A: 6 mg/kg body mass; Cohort B: 3 mg/kg body mass) 60 minutes before exercise.	Dietary supplement: Caffeine; Anhydrous caffeine administered orally in a hard gelatine capsule. To address the unique physiological profiles of the two cohorts, a highly specific, differentiated dosing protocol is implemented: Cohort A (adult functional fitness athletes) will receive a high-performance dose of 6 mg/kg body mass, while Cohort B (elite junior swimmers) will receive a safe, youth-adapted dose of 3 mg/kg body mass. In all experimental trials, the capsule is ingested exactly 60 minutes prior to the onset of the discipline-specific exercise test to match peak plasma caffeine concentration.; Dietary supplement: Dietary Nitrate (Beetroot Juice); Concentrated beetroot juice (BJ) administered as a single, acute organic shot of 70 mL (Beet It Sport, James White Drinks Ltd., Ipswich, UK). Each active shot contains approximately 400 mg (equivalent to ~6.4 mmol) of inorganic nitrate. In contrast to chronic loading designs, this study evaluates purely acute effects; the shot is ingested exactly 3 hours (180 minutes) prior to the physical performance test to align with peak systemic concentration of plasma nitrite.
Active Comparator: CAF + PLBJ (Caffeine + Placebo BJ); Participants ingest a nitrate-depleted beetroot juice shot (~0.04 mmol nitrates) 3 hours before exercise, and a capsule of caffeine (Cohort A: 6 mg/kg body mass; Cohort B: 3 mg/kg body mass) 60 minutes before exercise.	Dietary supplement: Caffeine; Anhydrous caffeine administered orally in a hard gelatine capsule. To address the unique physiological profiles of the two cohorts, a highly specific, differentiated dosing protocol is implemented: Cohort A (adult functional fitness athletes) will receive a high-performance dose of 6 mg/kg body mass, while Cohort B (elite junior swimmers) will receive a safe, youth-adapted dose of 3 mg/kg body mass. In all experimental trials, the capsule is ingested exactly 60 minutes prior to the onset of the discipline-specific exercise test to match peak plasma caffeine concentration.; Dietary supplement: Placebo (Nitrate-Depleted Beetroot Juice); Nitrate-depleted beetroot juice (Beet It, James White Drinks Ltd., Ipswich, UK) administered as a 70 mL shot. This placebo is identical in taste, smell, appearance, packaging, and viscosity to the active concentrated shot, but has been selectively filtered to contain negligible nitrate levels (<0.4 mmol / ~25 mg of nitrates). It is ingested exactly 3 hours prior to the exercise test.
Active Comparator: PLCAF + BJ (Placebo CAF + Beetroot Juice); Participants ingest a concentrated beetroot juice shot (~6.4 mmol / 400 mg nitrates) 3 hours before exercise, and a placebo capsule (maltodextrin) 60 minutes before exercise.	Dietary supplement: Dietary Nitrate (Beetroot Juice); Concentrated beetroot juice (BJ) administered as a single, acute organic shot of 70 mL (Beet It Sport, James White Drinks Ltd., Ipswich, UK). Each active shot contains approximately 400 mg (equivalent to ~6.4 mmol) of inorganic nitrate. In contrast to chronic loading designs, this study evaluates purely acute effects; the shot is ingested exactly 3 hours (180 minutes) prior to the physical performance test to align with peak systemic concentration of plasma nitrite.; Dietary supplement: Placebo (Maltodextrin Capsule); Placebo capsule containing inert maltodextrin powder. The capsule is identical in size, shape, weight, color (opaque), and texture to the active caffeine capsule to ensure robust blinding. It is administered at the exact same time point (60 minutes pre-exercise) according to the identical body-mass-adjusted dosing scheme (simulating either 3 mg/kg or 6 mg/kg).
Placebo Comparator: PLCAF + PLBJ (Double Placebo Control); Participants ingest a nitrate-depleted beetroot juice shot (~0.04 mmol nitrates) 3 hours before exercise, and a placebo capsule (maltodextrin) 60 minutes before exercise.	Dietary supplement: Placebo (Nitrate-Depleted Beetroot Juice); Nitrate-depleted beetroot juice (Beet It, James White Drinks Ltd., Ipswich, UK) administered as a 70 mL shot. This placebo is identical in taste, smell, appearance, packaging, and viscosity to the active concentrated shot, but has been selectively filtered to contain negligible nitrate levels (<0.4 mmol / ~25 mg of nitrates). It is ingested exactly 3 hours prior to the exercise test.; Dietary supplement: Placebo (Maltodextrin Capsule); Placebo capsule containing inert maltodextrin powder. The capsule is identical in size, shape, weight, color (opaque), and texture to the active caffeine capsule to ensure robust blinding. It is administered at the exact same time point (60 minutes pre-exercise) according to the identical body-mass-adjusted dosing scheme (simulating either 3 mg/kg or 6 mg/kg).

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Exercise Performance Time (minutes and seconds, Cohort A - Hyrox P'F'T)	Total time (in minutes and seconds) required to complete the standardized HYROX Physical Fitness Test (P'F'T) consisting of 6 specific functional exercises.	At each experimental visit (Visits T1-T4), with visits conducted at 7-day intervals following the baseline/familiarization visit (T0).
Swimming Performance (minutes and seconds, Cohort B - 8x100m Step-Test)	Swimming performance will be assessed using the completion time (seconds and milliseconds) for each 100-m repetition of the standardized 8 × 100-m step test, including the maximal-effort repetition performed during the fifth stage.	At each experimental visit (Visits T1-T4), with visits conducted at 7-day intervals following the baseline/familiarization visit (T0).

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Blood Lactate Kinetics (mmol/L)	Blood lactate concentration, including peak concentration, accumulation kinetics, and clearance rate, will be assessed from fingertip capillary blood collected before supplementation (baseline), pre-exercise (3 hours after beetroot juice supplementation and 30 minutes after caffeine supplementation), and post-exercise (3 and 7 minutes after exercise) at each experimental visit.	Before supplementation (baseline), pre-exercise (3 hours after beetroot juice supplementation and 30 minutes after caffeine supplementation), and 3 and 7 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Heart Rate (HR, bpm)	Real-time heart rate (beats per minute) will be continuously monitored throughout each exercise test using a telemetry-based heart rate monitoring system.	Continuously monitored during exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Rating of Perceived Exertion (RPE, points)	Subjective rating of perceived exertion will be assessed using the validated Borg Scale (6-20).	Immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Blood Pressure (mmHg)	Systolic and diastolic blood pressure will be measured using an automated oscillometric blood pressure monitor.	Measured 2.5 hours after beetroot juice supplementation and 30 minutes after caffeine supplementation (pre-exercise) at each experimental visit (Visits T1-T4; 7-day intervals).
Gastrointestinal Tolerance Profile (mVAS, points)	Gastrointestinal symptoms will be assessed using the modified Visual Analog Scale (mVAS; 0-10, where 0 = no symptoms and 10 = extremely severe symptoms).	Measured 30 minutes after supplementation and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
White blood cells count and differential (count/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
White blood cells count and differential (%)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (red blood cells, RBC) (count/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (hemoglobin concentration, HGB) (mmol/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (hematocrit, HCT) (L/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (mean corpuscular volume, MCV; mean platelet volume, MPV) (fL)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (mean corpuscular hemoglobin mass, MCH) (fmol)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (mean corpuscular hemoglobin concentration, MCHC) (mmol/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (platelet count, PLT) (count/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (platelet hematocrit, PCT) (cL/L)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (platelet distribution width, PDW; platelet large cell ratio, PLCR; red blood cells distribution width - coefficient of variation, RDW-C) (%)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Hematological indices (red blood cells distribution width - standard deviation, RDW-S) (fL)	Assessment of the hematological parameter will be performed from fingertip capillary blood collected before supplementation (baseline) and 3 minutes after exercise at each experimental visit.	Before supplementation (baseline) and 3 minutes after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Nutritional status indices (concentration of urea) (mmol/L)	Urea concentration will be assessed from fingertip capillary blood collected before supplementation (baseline).	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Creatinine Concentration (μmol/L)	Creatinine concentration will be assessed from fingertip capillary blood collected before supplementation (baseline).	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Muscle Damage Markers (ALT, AST, CK, LDH) (U/L)	Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) will be assessed from fingertip capillary blood collected before supplementation (baseline).	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Body composition (fat-free mass, fat mass) (kg)	Body composition will be assessed before supplementation (baseline) using bioelectrical impedance analysis.	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Body composition (fat-free mass, fat mass) (%)	Body composition will be assessed before supplementation (baseline) using bioelectrical impedance analysis.	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Body composition (total body water) (L)	Body composition will be assessed before supplementation (baseline) using bioelectrical impedance analysis.	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Body composition (total body water) (%)	Body composition will be assessed before supplementation (baseline) using bioelectrical impedance analysis.	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Iron Status (Iron, Ferritin, UIBC, TIBC) (μg/dL)	Serum iron concentration, ferritin concentration, unsaturated iron-binding capacity (UIBC), and total iron-binding capacity (TIBC) will be assessed from fingertip capillary blood collected before supplementation (baseline).	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Iron Status (Transferrin Concentration) (g/L)	Serum transferrin concentration will be assessed from fingertip capillary blood collected before supplementation (baseline).	Before supplementation (baseline) at each experimental visit (Visits T1-T4; 7-day intervals).
Sleep Quality (Visual Analog Scale, VAS; points)	Subjective sleep quality will be assessed using a Visual Analog Scale (VAS).	Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Mood (Brunel Mood Scale, BRUMS; points)	Mood state will be assessed using the Brunel Mood Scale (BRUMS).	Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Competitive State Anxiety (Revised Competitive State Anxiety Inventory-2, CSAI-2R; points)	Competitive state anxiety will be assessed using the Revised Competitive State Anxiety Inventory-2 (CSAI-2R).	Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Felt Arousal (Felt Arousal Scale, FAS; points)	Perceived arousal will be assessed using the Felt Arousal Scale (FAS).	Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
Feeling Scale (FS; points)	Affective valence will be assessed using the Feeling Scale (FS).	Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).

Collaborators and Investigators

• Sponsor: Poznan University of Physical Education
• Investigators: Principal Investigator: Natalia Główka, PhD, Poznan University of Physical Education, Department of Sports Dietetics

Study record dates

Study Major Dates

• Study Start (Estimated): October 1, 2026
• Primary Completion (Estimated): June 30, 2029
• Study Completion (Estimated): June 30, 2029

Study Registration Dates

• First Submitted: July 17, 2026
• First Submitted That Met QC Criteria: July 31, 2026
• First Posted (Actual): August 5, 2026

Study Record Updates

• Last Update Posted (Actual): August 7, 2026
• Last Update Submitted That Met QC Criteria: August 6, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Keywords: Nitric oxide; Caffeine; Sports nutrition; High-Intensity Functional Training; Swimming
• Additional Relevant MeSH Terms: Heterocyclic Compounds; Heterocyclic Compounds, 2-Ring; Heterocyclic Compounds, Fused-Ring; Alkaloids; Purinones; Purines; Xanthines; Caffeine; maltodextrin
• Other Study ID Numbers: ZDS2026_NG_001

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: The study protocol will be published as an open-access article. Upon completion of the study, de-identified individual participant data (IPD) and relevant analytic code will be deposited in an open-access repository such as Zenodo or OSF, following the FAIR principles.
• IPD Sharing Time Frame: The study protocol will be available upon its publication. The IPD and analytic code will be available starting from the date of the primary study results publication and will remain accessible for at least 5 years.
• IPD Sharing Access Criteria: The study protocol will be published as an open-access article. Upon completion of the study, de-identified individual participant data (IPD) and relevant analytic code will be deposited in an open-access repository such as Zenodo or OSF, in accordance with FAIR principles.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No