Clinical trial NCT05302804
Conditions: Acute Exercise Heat Exposure
Keywords: heat exposure menthol exercise
Sponsor: University of Nebraska
Start date: 2022-05-01; Completion date: 2022-10-01
Enrollment: 12 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this study is to examine the human thermoregulatory impact of applying a commercially available menthol gel (BioFreeze) to the skin prior to moderate intensity walking under heated conditions. Experimental Visits will consist of 30 min of treadmill walking at a moderate pace (3.5 mph, 5% grade) under hot conditions (38°C, 60%RH) and will be randomized and counterbalanced for BioFreeze or a hypoallergenic gel (control) application. Gels will be applied to areas commonly exposed during outdoor activity in warm conditions (shoulder to wrist, mid thigh to ankle). Accordingly, participants will wear shorts and a tank top shirt during exercise. Core temperature, skin temperature, galvanic skin response, laser doppler blood flow, and heart rate will be continuously recorded throughout the exercise bout via an integrated analog to digital converter. Sweat will be collected during exercise using commercially available absorbent patches. Thermal sensation will be assessed throughout exercise via the American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE) thermal sensation likert scale (cold to hot). Lastly, nude body weight will be recorded pre and post exercise for sweat rate determination. The BioFreeze and control trials will be separated by a 7-14 day washout period.

Detailed description:
3 experimental visits to the laboratory will be conducted. All visits will take place at the University of Nebraska at Omaha, Exercise Physiology Laboratory. Visit 1 will consist of the physical activity readiness questionnaire (PARQ), adverse reaction to BioFreeze, and body composition (hydrostatic weigh, bioelectrical impedance). Visits 2 and 3 will be separated by 7-14 days, randomized, and counterbalanced for experimental (BioFreeze) and control (hypoallergenic cream) trials. Both the experimental and control visits will follow the same protocol.

Participants will arrive following an overnight fast state while also refraining from strenuous activity, alcohol consumption, tobacco use, and recreational drugs for the previous 24 hour period. During these visit researchers will apply BioFreeze using a gloved hand from shoulder to wrist and mid thigh to ankle (areas outside of the clothing). A hypoallergenic cream will be applied during the control visit.

Exercise will consist of 30 min of moderate intensity treadmill walking (3.5 mph at 5% grade) within a heated temperature/humidity controlled chamber (38°C, 60% relative humidity). Participants will be weighed before, and after completion of the exercise calculate sweat rate. Prior to the exercise session, participants will sit for 5 min in an ambient room temperature environment as a baseline. During baseline and during the exercise core temperature, skin temperature, laser doppler blood flow, galvanic skin response, and heart rate will be collected continuously. Sweat patches will be adhered to the forehead during exercise at minute 10 for collection of sweat composition over the next 10-20 minutes. Thermal perception using the ASHRAE scale (cold to hot) will be assessed pre and during exercise.

VISIT 1 Visit 1 will take approximately 1 hour. Informed consent will be given. Participants will then complete the physical activity readiness questionnaire (PARQ) to be cleared for physical activity. Participants will have a small amount of the BioFreeze cream applied to a small area of the forearm (\~2 x 2 in) to test for any adverse reaction over a period of 15 minutes. This initial skin reaction test is not a part of the research protocol. Those without a reaction will have height, weight, and body fat composition measured. Height and weight will be measured using a medical scale and stadiometer, respectively. Body fat will be assessed with hydrostatic weighing using an electronic load cell based system (Exertech, Dresbach, MN) correcting for residual lung volume or via a bio-electrical impedance analyzer (InBodyUSA, Cerritos, CA).

VISITS 2 AND 3 Visits 2 and 3 will take approximately 2 hours each. Upon arrival at the lab, a nude body weight will be collected with the subject in a private room. While still in the room, subjects will self-insert a rectal thermistor 12-15 cm beyond the anal sphincter and don a chest strap heart rate monitor. After dressing in their exercise clothing, subjects will exit the private room so that chest, forearm, and calf thermistors can be adhered to the skin with tape. Laser doppler flow (forearm and or/finger) will be adhered with tape and galvanic skin response (fingers) will be adhered using Velcro straps to the skin. Measuring instruments will be recorded using an integrated digital to analog converter (ADInstruments, Colorado Springs, CO).

Following instrumentation, subjects will sit for 5 minutes as a baseline in a temperate environment. Immediately after, a researcher with gloved hands will topically apply BioFreeze or the control hypoallergenic cream from the shoulder to wrist and mid-thigh to ankle. Subjects will then enter the heat chamber (38°C, 60% relative humidity) and immediately begin a 30-minute walk on a treadmill (3.5 mph, 5% grade). Subjects will be intermittently asked to hold their arm steady for laser doppler flow collection (10, 20, 30 minutes). Subjects will also briefly pause at minute 10 in order to adhere an absorbent patch to their forehead for sweat collection over the following 10-20 minutes. Thermal perception using the ASHRAE scale (cold to hot) will be assessed pre and during exercise. After 30 minutes of walking, subjects will exit the chamber for removal of all skin and finger sensors. Subjects will then re-enter the private room, remove their own rectal thermistor with gloved hands, and then a final nude body weight will be recorded after toweling off.

The rectal thermistor will measure core temperature. The heart rate monitor will measure the beats of the heart. Skin thermistors will measure the temperature of the skin surfaces. Laser doppler flow measure the relative units of blood flow velocity. Galvanic skin response measures skin conductivity changes as individuals begin to sweat. Sweat is collected for assessment of sweat composition. Thermal perception is assessed for individual perception surrounding the temperature of the ambient environment. Nude body weight is measured to calculate sweat rate.

Arms: arm 1: Participants randomized to receive menthol gel first then hypoallergenic gel after 7 day washout.

Gel is applied to the skin (arm and thigh). Participants walk on a treadmill for 30 minutes (3.5 mph at 5% grade) in 38°C and 60% relative humidity. arm 2: Participants randomized to receive hypoallergenic gel first then menthol gel after 7 day washout.

Gel is applied to the skin (arm and thigh). Participants walk on a treadmill for 30 minutes (3.5 mph at 5% grade) in 38°C and 60% relative humidity.
Interventions: intervention 1: Menthol Gel intervention 2: Control Gel
Intervention descriptions: intervention 1: 5% menthol gel applied from shoulder to wrist and mid thigh to ankle, 30 minute treadmill walk (3.5 mph at 5% grade at 38°C, 60% relative humidity) intervention 2: Hypoallergenic gel applied from shoulder to wrist and mid thigh to ankle, 30 minute treadmill walk (3.5 mph at 5% grade at 38°C, 60% relative humidity)

Protocol documents:
Prot_SAP_001.pdf: 
Study Protocol and Statistical Analysis Plan 
 
Title: The Influence of Topical Menthol Gel on Thermoregulatory and Perceptual Outcomes During Exercise 
Within the Heat  
NCT#: 05302804  
Unique Protocol ID 0111-22-FB 
Date: 21 Apr 2022 
 
 
 


Abstract 
The purpose of this study is to examine the human thermoregulatory impact of applying a 
commercially available menthol gel (BioFreeze) to the skin prior to moderate intensity walking 
under heated conditions. Eligible participants will be aged 19-45 years, have no previously 
known adverse reactions to menthol (active ingredient in BioFreeze) based upon a self-report, 
no adverse reactions to BioFreeze as determined through gel application to a small forearm 
area, and cleared for physical activity via the 2022 Physical Activity Readiness Questionnaire 
(PARQ). There will be a total of 3 experimental visits to the laboratory. Visit 1 will test for 
adverse BioFreeze reaction by applying a small amount of gel to the forearm (~2 x 2 in). 
Anthropometric and body composition data will also be collected at this time. Visits 2 and 3 
will consist of 30 min of treadmill walking at a moderate pace (3.5 mph, 5% grade) under hot 
conditions (38°C, 60%RH). Visits 2 and 3 will be randomized and counterbalanced for 
BioFreeze or a hypoallergenic gel (control) application. Gels will be applied to areas 
commonly exposed during outdoor activity in warm conditions (shoulder to wrist, mid-thigh to 
ankle). Accordingly, participants will wear shorts and a tank top shirt during exercise. Core 
temperature, skin temperature, galvanic skin response, laser doppler blood flow, and heart 
rate will be continuously recorded throughout the exercise bout via an integrated analog to 
digital converter. Sweat will be collected during exercise using commercially available 
absorbent patches. Thermal sensation will be assessed throughout exercise via the ASHRAE 
thermal sensation likert scale (cold to hot). Lastly, nude body weight will be recorded pre and 
post exercise for sweat rate determination. The BioFreeze and control trials will be separated 
by a 7 to 14 day washout period.  
 
Purpose and Rationale 
The purpose of this study is to examine the impact of applying a menthol-based gel to the skin 
on human thermoregulation and thermal perception during exercise in the heat. 
Transient receptor potential (TRP) channels are a family of membrane proteins evolved to 
interpret environmental stimuli through ion channels. One of these channels, the TRP 
melastatin 8 (TRPM8), is responsible for the sensation of innocuous cool temperatures and it 
might be responsible for noxious cold temperatures [1,2]. When applied topically, menthol 
acts upon a cold transduction enzyme to decrease the thermal sensitivity of TRPM8. This 
decreased thermal sensitivity allows activation of TRPM8 at warmer temperatures, mimicking 
a cool sensation in warm environments. This cool menthol feeling is used widely in everyday 
commercial products, but most popular when used as an analgesic in medicated pain-
relieving gels/creams. 
Current recommendations suggest menthol can be used as an ergogenic aid resulting in an 
improved performance within heated environments [1]. By decreasing thermal sensitivity of 
TRPM8 receptors, menthol exerts a physiological response that is perceived as external 
cooling. The most salient response is an increase in heat conservation, resulting in a reduced 
exercise capacity due to fatigue associated with a rise in core body temperature [3]. It seems 


the application and concentration strength of menthol application to the skin may be the most 
obvious influential factors. As such, researchers have used proprietary menthol solutions with 
varying intensities (0.05- 10%) [1], yet, to our knowledge, no one has investigated widely 
available, over-the- counter products. Indeed, the translation from proprietary formula to over-
the-counter application is unknown. Furthermore, it is unclear if currently available menthol 
products may also aid in performance. This incongruence requires further investigation into 
the effect of menthol on exercise performance. 
Participants 
12-15 subjects, age 19 to 45 years, will be needed to complete the study. Nineteen is the age 
of majority at the study site. The 2022 physical activity readiness questionnaire (PARQ) 
requires that adults aged >45 who are not accustomed to regular physical activity seek 
professional consultation prior to beginning physical activity. Thereby, keeping an age range 
of 19-45 ensures that the participants will be of consenting age and cleared for physical 
activity participation. 
Research Plan 
Three experimental visits to the laboratory will be conducted. All visits will take place at the 
University of Nebraska at Omaha, Exercise Physiology Laboratory. Visit 1 will consist of the 
physical activity readiness questionnaire (PARQ), adverse reaction to BioFreeze, and body 
composition (hydrostatic weigh, bioelectrical impedance). Visits 2 and 3 will be separated by 
7-14 days, randomized, and counterbalanced for experimental (BioFreeze) and control 
(hypoallergenic cream) trials. Both the experimental and control visits will follow the same 
protocol. 
Participants will arrive following an overnight fast state while also refraining from strenuous 
activity, alcohol consumption, tobacco use, and recreational drugs for the previous 24-hour 
period. During these visit researchers will apply BioFreeze using a gloved hand from shoulder 
to wrist and mid-thigh to ankle (areas outside of the clothing). A hypoallergenic cream will be 
applied during the control visit. 
Exercise will consist of 30 min of moderate intensity treadmill walking (3.5 mph at 5% grade) 
within a heated temperature/humidity-controlled chamber (38°C, 60% relative humidity). 
Participants will be weighed before, and after completion of the exercise calculate sweat rate. 
Prior to the exercise session, participants will sit for 5 min in an ambient room temperature 
environment as a baseline. During baseline and during the exercise core temperature, skin 
temperature, laser doppler blood flow, galvanic skin response, and heart rate will be collected 
continuously. Sweat patches will be adhered to the forehead during exercise at minute 10 for 
collection of sweat composition over the next 10-20 minutes. 
Thermal perception using the ASHRAE scale (cold to hot) will be assessed pre and during 
exercise. 
 


VISIT 1 
Visit 1 will take approximately 1 hour. Informed consent will be given. Participants will then 
complete the physical activity readiness questionnaire (PARQ) to be cleared for physical 
activity. Participants will have a small amount of the BioFreeze cream applied to a small area 
of the forearm (~2 x 2 in) to test for any adverse reaction over a period of 15 minutes. This 
initial skin reaction test is not a part of the research protocol. Then, resting blood pressure will 
be taken. Those without a reaction, and a resting blood pressure lower than 140/90, will have 
height, weight, and body fat composition measured. Height and weight will be measured using 
a medical scale and stadiometer, respectively. Body fat will be assessed with hydrostatic 
weighing using an electronic load cell-based system (Exertech, Dresbach, MN) correcting for 
residual lung volume or via a bio-electrical impedance analyzer (InBodyUSA, Cerritos, CA). 
 
VISITS 2 AND 3 
Visits 2 and 3 will take approximately 2 hours each. Upon arrival at the lab, a nude body 
weight will be collected with the subject in a private room. While still in the room, subjects will 
self-insert a rectal thermistor 12-15 cm beyond the anal sphincter and don a chest strap heart 
rate monitor. After dressing in their exercise clothing, subjects will exit the private room so that 
chest, forearm, and calf thermistors can be adhered to the skin with tape. 
Laser doppler flow (forearm and or/finger) will be adhered with tape and galvanic skin 
response (fingers) will be adhered using Velcro straps to the skin. Measuring instruments will 
be recorded using an integrated digital to analog converter (ADInstruments, Colorado 
Springs, CO). 
Following instrumentation, subjects will sit for 5 minutes as a baseline in a temperate 
environment. Immediately after, a researcher with gloved hands will topically apply BioFreeze 
or the control hypoallergenic cream from the shoulder to wrist and mid-thigh to ankle. 
Subjects will then enter the heat chamber (38°C, 60% relative humidity) and immediately 
begin a 30-minute walk on a treadmill (3.5 mph, 5% grade). Subjects will be intermittently 
asked to hold their arm steady for laser doppler flow collection (10, 20, 30 minutes). Subjects 
will also briefly pause at minute 10 in order to adhere an absorbent patch to their forehead for 
sweat collection over the following 10-20 minutes. Thermal perception using the ASHRAE 
scale (cold to hot) will be assessed pre and during exercise. After 30 minutes of walking, 
subjects will exit the chamber for removal of all skin and finger sensors. Subjects will then re-
enter the private room, remove their own rectal thermistor with gloved hands, and then a final 
nude body weight will be recorded after toweling off. 
The rectal thermistor will measure core temperature. The heart rate monitor will measure the 
beats of the heart. Skin thermistors will measure the temperature of the skin surfaces. Laser 
doppler flow measure the relative units of blood flow velocity. 
Galvanic skin response measures skin conductivity changes as individuals begin to sweat. 
Sweat is collected for assessment of sweat composition. Thermal perception is assessed for 


individual perception surrounding the temperature of the ambient environment. Nude body 
weight is measured to calculate sweat rate. 
 
Statistical Analysis 
Recruitment 
Previous exercise research suggests that the difference in response of matched pairs is 
normally distributed with a standard deviation of 0.86. If the true difference of the mean 
response of matched pairs is 0.75, we will need to complete 12 experimental trials for each 
condition to be able to reject the null hypothesis that this response difference is zero with a 
power of 0.8. The Type I error probability associated with this test of the null hypothesis is 
0.05. 
Interventions 
A repeated measures two-way ANOVA (Time x condition) will be used for the dependent 
variables. If the F-ratio values are found to be significant, a Fisher's Least Significant 
Difference post hoc analysis will be conducted. A type I error probability of < 5% will be 
considered significant (p< 0.05). All data will be analyzed using a combination of Microsoft 
Excel and the Statistical Package for the Social Sciences (SPSS).