Betaine Addition to Water Loading Prior to Running Exercise

Effects of a Betaine-Supplemented Hydration Protocol on Sweat Rate and Exercise Performance in Runners

This randomized, double-blind, placebo-controlled crossover study will investigate whether acute betaine ingestion as part of a pre-exercise hyperhydration strategy improves endurance running performance and influences hydration-related responses during prolonged exercise.

Approximately 30 recreationally trained male and premenopausal female runners will complete two experimental trials: a betaine condition and a placebo/control condition. In the betaine condition, participants will ingest anhydrous betaine at a dose of 0.07 g/kg body mass as part of a standardized pre-exercise hydration protocol providing a total fluid intake of approximately 25 mL/kg body mass.

Following the hydration protocol, participants will complete 60 minutes of treadmill running at 60% of the running velocity corresponding to VO₂max, followed by a 5-minute recovery period and a 5-km treadmill time trial.

The primary outcome will be 5-km running time-trial performance. Secondary outcomes will include exercise-induced fluid and sweat loss, urinary hydration markers, sweat electrolyte concentrations, physiological responses, exercise economy, substrate utilization, blood lactate concentration, perceived exertion, thirst, and thermal sensation.

Study Overview

• Status: Not yet recruiting
• Conditions: Exercise; Hydration
• Intervention / Treatment: Dietary supplement: Anhydrous Betaine; Other: Placebo

• Study Type: Interventional
• Enrollment (Estimated): 20
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Emilia Zawieja; Phone Number: +48500415726; Email: emilia.zawieja@up.poznan.pl

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 45 years (Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

Description

Inclusion Criteria:

• Age 18-45 years
• Sex: men and premenopausal women
• Continuous running training for at least 12 months
• At least 3 running training sessions per week
• Willingness and ability to complete the treadmill exercise protocol: 60 minutes of running at 60% of the running velocity corresponding to VO₂max, followed by a 5-km running time trial
• Medical clearance for participation in maximal and prolonged exercise testing

Exclusion Criteria:

• Age <18 or >45 years
• Postmenopausal women
• Use of betaine, choline, folic acid/folate, or vitamin B12 supplements within 3 months prior to study participation
• Current musculoskeletal injury or injury occurring within the previous 2 months
• Uncontrolled arterial hypertension
• Pregnancy or breastfeeding
• Diabetes requiring insulin therapy or inadequately controlled diabetes
• Active or severe liver disease, including ALT or AST >3× the upper limit of normal
• Renal impairment, including eGFR <60 mL/min/1.73 m²
• Untreated or unstable thyroid disease, including hypothyroidism or hyperthyroidism
• Gastrointestinal disease that may affect nutrient absorption
• Kidney disease, cardiovascular disease, irritable bowel syndrome, or adrenal disease
• Use of diuretics, SGLT2 inhibitors, laxatives, or glucocorticoids
• Any medical contraindication to physical exercise and/or failure to obtain medical clearance for exercise testing
• Elevated body temperature at the time of testing
• Cystic fibrosis

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Other
• Allocation: Randomized
• Interventional Model: Crossover Assignment
• Masking: Triple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Betaine → Placebo Sequence; Participants will first complete the betaine-enriched hyperhydration condition. After the washout period, they will complete the placebo hyperhydration condition.	Dietary supplement: Anhydrous Betaine; Anhydrous betaine (trimethylglycine) will be administered as a single dose of 0.07 g/kg body mass, dissolved in a commercially available sports drink (5 mL/kg body mass), 150 minutes before exercise. The betaine-containing drink will constitute the first bolus of a standardized pre-exercise hyperhydration protocol providing a total fluid intake of approximately 25 mL/kg body mass over 120 minutes.; Other: Placebo; Participants will receive the same commercially available sports drink (5 mL/kg body mass) without added betaine 150 minutes before exercise. This will be followed by four additional fluid boluses of mineral water (5 mL/kg each) administered at 120, 90, 60, and 30 minutes before exercise, for a total pre-exercise fluid intake of approximately 25 mL/kg body mass.
Experimental: Placebo → Betaine Sequence; Participants will first complete the placebo hyperhydration condition. After the washout period, they will complete the betaine-enriched hyperhydration condition.	Dietary supplement: Anhydrous Betaine; Anhydrous betaine (trimethylglycine) will be administered as a single dose of 0.07 g/kg body mass, dissolved in a commercially available sports drink (5 mL/kg body mass), 150 minutes before exercise. The betaine-containing drink will constitute the first bolus of a standardized pre-exercise hyperhydration protocol providing a total fluid intake of approximately 25 mL/kg body mass over 120 minutes.; Other: Placebo; Participants will receive the same commercially available sports drink (5 mL/kg body mass) without added betaine 150 minutes before exercise. This will be followed by four additional fluid boluses of mineral water (5 mL/kg each) administered at 120, 90, 60, and 30 minutes before exercise, for a total pre-exercise fluid intake of approximately 25 mL/kg body mass.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
5-km Running Time-Trial Performance	Time required to complete a 5-km treadmill running time trial, expressed in seconds. Participants will be instructed to complete the distance as quickly as possible and will be allowed to adjust treadmill speed throughout the trial. Lower completion time indicates better performance.	During the 5-km treadmill time trial performed after the 60-minute running preload and a 5-minute recovery period in each experimental trial.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Sweat rate	Whole-body sweat rate will be estimated from the change in body mass from immediately before exercise to completion of the 5-km time trial, adjusted for fluid intake and urine output, if applicable, and expressed as L/h. Body mass will be measured using the calibrated electronic scale integrated with the BOD POD system (COSMED, Italy) and expressed in kilograms (kg). Changes in body mass will also be calculated and reported as a percentage of pre-exercise body mass (%).	From immediately before the 60-minute running preload to completion of the subsequent 5-km time trial during each experimental trial.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Sweat sodium concentration	Sodium concentration will be measured in sweat samples collected from standardized sites on the forearm and thigh during exercise. Sweat samples will be collected between minutes 30 and 60 of the 60-minute running preload. Sodium concentration will be analyzed using a HORIBA ion-selective electrode analyzer and reported in mmol/L.	Between minutes 30 and 60 of the 60-minute running preload during each experimental trial.
Sweat Potassium Concentration	Potassium concentration will be measured in sweat samples collected from standardized sites on the forearm and thigh during exercise. Sweat samples will be collected between minutes 30 and 60 of the 60-minute running preload. Sweat potassium concentration will be analyzed using a HORIBA ion-selective electrode analyzer and reported in mmol/L.	Between minutes 30 and 60 of the 60-minute running preload during each experimental trial.
Blood lactate concentration	Blood lactate concentration will be measured using a portable Lactate Scout 4 analyzer. Measurements will be obtained immediately after completion of the 60-minute running preload and immediately after completion of the subsequent 5-km time trial. Blood lactate concentration will be reported in mmol/L.	Immediately after the 60-minute running preload and immediately after the 5-km time trial during each experimental trial.
Heart rate	Heart rate will be monitored continuously using a Garmin chest-strap heart rate monitor with integrated electrodes throughout the 60-minute running preload and the subsequent 5-km time trial. Outcome variables will include mean heart rate during the 60-minute preload, mean heart rate during the 5-km time trial, and maximal heart rate achieved during the 5-km time trial. Heart rate will be reported in beats per minute (bpm).	Continuously during the 60-minute running preload and the subsequent 5-km time trial in each experimental trial.
Core Body Temperature	Core body temperature will be monitored continuously using a CORE body temperature sensor attached to the Garmin chest strap throughout the 60-minute running preload and the subsequent 5-km time trial. Outcome variables will include mean core body temperature during the exercise protocol and the highest core body temperature recorded during the exercise protocol. Temperature will be reported in degrees Celsius (°C).	Continuously during the 60-minute running preload and the subsequent 5-km time trial in each experimental trial.
Running Economy	Running economy will be assessed by breath-by-breath indirect calorimetry using a QUARK RMR metabolic system during the final 10 minutes (minutes 50-60) of the 60-minute running preload. Running economy will be quantified as mean oxygen consumption (VO₂) relative to body mass at the standardized running velocity and expressed as mL·kg-¹·min-¹. Lower oxygen consumption at the same running velocity indicates better running economy.	During the final 10 minutes (minutes 50-60) of the 60-minute running preload in each experimental trial.
Respiratory Exchange Ratio	Respiratory exchange ratio (RER) will be assessed by breath-by-breath indirect calorimetry using a QUARK RMR metabolic system during the final 10 minutes (minutes 50-60) of the 60-minute running preload. RER will be calculated as the ratio of carbon dioxide production (VCO₂) to oxygen consumption (VO₂) and expressed as a unitless value. The mean RER over the measurement period will be used for analysis.	During the final 10 minutes (minutes 50-60) of the 60-minute running preload in each experimental trial.
Urine specific gravity	Urine specific gravity (USG) will be measured using a URIT-31 urine analyzer from urine samples collected immediately before the 60-minute running preload and after completion of the subsequent 5-km time trial. USG will be reported as a dimensionless value. Urine osmolality will also be estimated from USG using a predefined conversion equation and reported in mOsm/kg.	Immediately before the 60-minute running preload and immediately after completion of the 5-km time trial in each experimental trial.
Rating of Perceived Exertion (RPE)	Rating of perceived exertion will be assessed using the Borg 6-20 Rating of Perceived Exertion scale, ranging from 6 ("no exertion at all") to 20 ("maximal exertion"). RPE will be recorded at 15-minute intervals during the 60-minute running preload and immediately after completion of the 5-km time trial.	At minutes 30, and 60 of the 60-minute running preload and immediately after completion of the 5-km time trial during each experimental trial.
Thirst Sensation	Perceived thirst will be assessed using a 9-point categorical thirst sensation scale ranging from 1 ("not thirsty at all") to 9 ("very, very thirsty"). Participants will be asked to rate their current sensation of thirst at each measurement time point.	Immediately before the 60-minute running preload, at minutes 30,and 60 of the preload, and immediately after completion of the 5-km time trial during each experimental trial.
Thermal Sensation	Thermal sensation will be assessed using a 17-point thermal sensation scale ranging from 0 to 8 in 0.5-point increments, with 0 representing "unbearably cold", 4 representing "neutral", and 8 representing "unbearably hot". Participants will rate their whole-body thermal sensation at each measurement time point.	Immediately before the 60-minute running preload, at minutes 30 and 60 of the preload, and immediately after completion of the 5-km time trial during each experimental trial.

Collaborators and Investigators

• Sponsor: Poznan University of Life Sciences

Study record dates

Study Major Dates

• Study Start (Estimated): October 1, 2026
• Primary Completion (Estimated): May 31, 2027
• Study Completion (Estimated): December 30, 2027

Study Registration Dates

• First Submitted: September 18, 2026
• First Submitted That Met QC Criteria: September 18, 2026
• First Posted (Actual): September 25, 2026

Study Record Updates

• Last Update Posted (Actual): September 25, 2026
• Last Update Submitted That Met QC Criteria: September 18, 2026
• Last Verified: September 1, 2026

More Information

Terms related to this study

• Keywords: exercise; endurance; betaine; hydration
• Additional Relevant MeSH Terms: Behavior; Motor Activity
• Other Study ID Numbers: BETxSWEAT

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

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