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.
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
Allocation
RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
QUADRUPLE
Enrollment
112
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.
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.
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).
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.
Poznan University of Physical Education
Poznan, Wielkopolska, Poland
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.
Time frame: 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.
Time frame: At each experimental visit (Visits T1-T4), with visits conducted at 7-day intervals following the baseline/familiarization visit (T0).
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.
Time frame: 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.
Time frame: 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).
Time frame: 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.
Time frame: 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).
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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.
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: 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).
Time frame: Before supplementation (baseline), pre-exercise, and immediately after exercise at each experimental visit (Visits T1-T4; 7-day intervals).
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