Acute Effects of Menthol Gargle and Buccal Film on Anaerobic Capacity and Respiratory Parameters in Male Volleyball Players

BACKGROUND AND RATIONALE Volleyball is an intermittent team sport characterized by repeated jumps, sudden accelerations, short-duration maximal or near-maximal movements, and limited recovery intervals. Rapid lower-extremity power production and the ability to sustain it across repeated high-intensity actions are therefore central to volleyball performance.

Menthol is a natural monoterpene that produces a cooling sensation by activating the TRPM8 channel in thermosensory neurons. This sensory response does not correspond to an actual reduction in skin or core temperature, which suggests that any acute effect of oral menthol on exercise may operate through perceptual and behavioral rather than thermal mechanisms.

Findings on strength, power and high-intensity exercise are more limited and inconsistent than those on thermal perception, and recent meta-analytic evidence does not support menthol mouth-rinsing as a consistent ergogenic strategy. Whether menthol affects anaerobic performance under thermoneutral conditions during short-duration maximal exercise remains unclear.

In most exercise studies menthol has been delivered as a mouth rinse, beverage, gel or topical preparation. A buccal film is a distinct oral dosage form: it adheres to the buccal mucosa and, depending on formulation, can alter the residence time and release profile of the incorporated compound in the oral cavity. In pharmaceutical research menthol has generally been studied as a permeation enhancer or excipient rather than as the primary active compound. The dissolution rate, release profile, mucosal absorption and sensory exposure produced by a menthol-containing buccal film cannot be inferred from mouth-rinsing studies. The buccal film is therefore investigated here as an experimental route of administration with no established physiological or ergogenic superiority, not as a presumed enhancement.

HIF-1alpha regulates genes associated with glycolytic metabolism, oxygen homeostasis and angiogenesis, and exercise has been shown to affect HIF-1alpha protein levels and HIF-1-related signaling in human skeletal muscle. At the cell-culture level, menthol-induced TRPM8 activation has been reported to stabilize HIF-1alpha protein through a calcium- dependent, oxygen-independent mechanism. HIF-1alpha is therefore included as an exploratory biomarker of the biological response to high-intensity exercise under the three conditions.

OBJECTIVES Primary objective: to compare the acute effects of placebo (water control), menthol mouth-rinsing, and a menthol-containing buccal film on anaerobic performance assessed by the Wingate Anaerobic Power Test in male volleyball players. Secondary (exploratory) objectives: to compare the effects of the three conditions on respiratory function, post-exercise blood lactate concentration, and post-exercise plasma HIF-1alpha concentration.

HYPOTHESIS There will be significant differences among the placebo, menthol mouth-rinsing, and menthol-containing buccal-film conditions in anaerobic performance, respiratory function, blood lactate concentration, and plasma HIF-1alpha concentration.

DESIGN Single-blind, randomized, three-condition crossover trial. Each participant completes all three conditions. The order of conditions is determined by randomization. A 72-hour washout period separates conditions to minimize carryover effects.

SAMPLE SIZE An a priori power analysis was conducted using G*Power 3.1.9.7 for a repeated-measures within-subjects ANOVA with one group and three repeated measurements, assuming an effect size f = 0.35, alpha = 0.05, power (1-beta) = 0.80, correlation among repeated measures r = 0.50, and nonsphericity correction epsilon = 1.00. The required minimum sample size was 15 participants (achieved power 0.81). Nineteen volunteers were assessed for eligibility to allow for attrition.

INTERVENTIONS Placebo (water control): a total of 40 mL of tap water given as two separate 20 mL volumes; each volume is rinsed around the mouth for 30 seconds and expectorated without swallowing (total rinsing time 60 seconds). Menthol gargle (MG): a total of 40 mL of solution containing 10 mg L-menthol (0.025% w/v), given as two separate 20 mL volumes; each volume is rinsed for 30 seconds and expectorated without swallowing (total rinsing time 60 seconds). The concentration was selected based on the range of L-menthol concentrations used in exercise studies. Menthol buccal film (MBF): a film containing a total of 10 mg L-menthol placed on the buccal mucosa within the oral cavity. Total L-menthol content is standardized at 10 mg across both active conditions.

PROCEDURES PER SESSION

1. Body weight is remeasured to set the Wingate resistance load.
2. Standardized 15-minute warm-up (10 minutes light running, 5 minutes stretching).
3. Administration of the assigned condition (P, MG or MBF).
4. Spirometry in the seated position, with nose clip and disposable mouthpiece, performed according to the 2019 ATS/ERS spirometry standardization statement. At least three acceptable maneuvers are obtained and FEV1/FVC repeatability is verified. The highest FVC and FEV1 from acceptable maneuvers are used.
5. Approximately one minute after spirometry, the 30-second Wingate Anaerobic Power Test is performed on a mechanically braked Monark 834E cycle ergometer at a resistance of 7.5% of body weight, with standardized verbal encouragement, participants remaining seated.
6. Immediately after the Wingate test, a 5 mL venous blood sample is drawn from the antecubital vein into an EDTA tube.

BLOOD SAMPLE HANDLING Tubes are inverted 8-10 times. Within 30 minutes of collection, samples are centrifuged at 1500 x g for 15 minutes at 4 degrees C. Plasma is aliquoted into sterile microtubes without contacting the cellular layer and stored at -80 degrees C until analysis.

BIOCHEMICAL ANALYSES Plasma HIF-1alpha is measured by a commercial human HIF-1alpha sandwich ELISA (Reed Biotech Ltd., Wuhan, China; catalogue no. RE2787H) and expressed as pg/mL. Lactate is measured by a commercial lactic acid assay kit (Reed Biotech Ltd., Wuhan, China; catalogue no. RE10064) and expressed as mmol/L. Samples and standards are analyzed in duplicate; absorbance is read on a BioTek ELx800 microplate reader.

STANDARDIZATION AND CONTROL OF CONFOUNDERS Participants refrain from menthol-containing products for one week before the first condition, and from caffeine, alcohol, menthol-containing products and strenuous exercise for 72 hours before each condition. Dietary intake is recorded for the 72 hours before the first condition and participants are asked to maintain a similar dietary pattern before subsequent conditions. All sessions are conducted at 72-hour intervals, at the same time of day, under similar environmental conditions, and by the same researcher. Ergometer seat and handlebar settings are individualized at the first session and kept unchanged thereafter.

BLINDING Participants are not informed of the study hypothesis or the expected direction of effects.

The researcher preparing and administering the conditions is aware of the allocation.

Because of the differences between routes of administration and the perceptible cooling sensation produced by menthol, complete participant blinding is not achievable; blinding is therefore implemented by restricting participants' access to information about the hypothesis and expected effects.

STATISTICAL ANALYSIS Continuous variables are summarized as mean and standard deviation. Normality is assessed with the Shapiro-Wilk test. Differences among conditions are examined with one-way repeated-measures ANOVA with condition as a three-level within-subject factor. Sphericity is assessed with Mauchly's test. For variables with a significant omnibus condition effect, pairwise comparisons are performed with Bonferroni correction. Effect size is reported as partial eta squared. All tests are two-tailed with alpha set at 0.05.