Study Design: Randomized Cross-Over Study

Objective:

This study aims to compare the acute effects of three different auditory conditions (metronome, binaural beats, and silent control) on 6-Minute Walk Test (6MWT) performance, post-exercise recovery physiology, and perceived exertion in healthy young adults.

Background:

Auditory-motor coupling mechanisms suggest that rhythmic auditory stimuli can lower the excitation threshold of motor neurons, thereby enhancing walking efficiency. Rhythmic stimuli such as metronomes are known to improve walking speed and coordination. Binaural beats are an auditory stimulus type generated by presenting two tones of slightly different frequencies to each ear, triggering cortical neural entrainment. It has been reported that listening to binaural beats at theta frequency following exercise increases parasympathetic activation and accelerates recovery. This study aims to examine these two auditory stimuli in comparison with a control condition in healthy young adults.

Participants:

Healthy young adults aged 18-35 who can walk independently and have no cardiovascular, pulmonary, neurological, or musculoskeletal conditions. Minimum sample size will be calculated using G*Power following a pilot study, with a 20% dropout margin added.

Randomization:

All participants will be exposed to all three conditions on separate days. The order of application will be determined by computer-based simple randomization (randomizer.org). Six possible sequence combinations will be used to balance order effects. A washout period of at least 24 hours will be applied between sessions.

Interventions:

Metronome Condition: Each participant's natural walking cadence will be measured, and the metronome tempo will be set at 110% of this baseline value.

Binaural Beats Condition: A carrier frequency of 120 Hz will be delivered to the right ear and 100 Hz to the left ear, generating a 20 Hz binaural beat in the Beta frequency band. Participants will listen for 6 minutes at rest immediately before the walking test.

Control Condition: No auditory stimulus will be applied.

The 6MWT will be conducted in all conditions along a 30-meter flat corridor in accordance with ATS/ERS standards.

Measurement Time Points: Pre-test, immediately post-test, and at the 1st, 3rd, and 5th minutes of the recovery period.

Outcome Measures:

Primary: Total distance covered during 6MWT (meters), walking cadence (steps/min) Secondary: Heart rate, blood pressure, peripheral oxygen saturation (SpO₂), perceived dyspnea, general fatigue, and quadriceps femoris muscle fatigue assessed via the Modified Borg Scale

Statistical Analysis:

One-Way Repeated Measures ANOVA for walking distance and cadence; Two-Way Repeated Measures ANOVA (condition × time) for physiological parameters; Friedman test for Borg scale data; Bonferroni-corrected post-hoc tests where significant differences are found. Significance level set at p<0.05.

Study Duration: April - June 2026 (3 months)

This study employs a randomized cross-over design in which each participant will undergo all three auditory conditions (binaural beats, metronome, and silent control) on separate days. To minimize potential carryover effects from a preceding condition, a washout period of at least 24 hours will be maintained between sessions. The order of condition administration will be determined by computer-assisted simple randomization (https://www.randomizer.org/) to balance potential order, fatigue, and learning effects. Participants will be randomly assigned to one of six possible sequences:

Sequence 1: Control - Metronome - Binaural Sequence 2: Control - Binaural - Metronome Sequence 3: Metronome - Control - Binaural Sequence 4: Metronome - Binaural - Control Sequence 5: Binaural - Control - Metronome Sequence 6: Binaural - Metronome - Control

Interventions

1. Metronome Condition (Rhythmic Auditory Stimulation) To objectively determine each participant's natural walking cadence, a 30-second pre-walk assessment will be conducted before the test session. The participant will be asked to walk at their preferred comfortable pace, and the number of steps taken over 30 seconds will be counted and recorded by the researcher. This value will be multiplied by two to calculate the 1-minute baseline walking cadence (steps/minute) (Chase et al., 2023). The metronome tempo accompanying the test will then be standardized at 110% of the participant's baseline cadence (i.e., 10% above their natural rhythm), in accordance with the protocol established by Yu et al. (2015). The target metronome rhythm (beats per minute, BPM) will be calculated by multiplying the baseline cadence by a coefficient of 1.10. The metronome stimulus will be delivered continuously throughout the 6MWT via open-ear headphones.

2. Binaural Beats Condition Binaural beats stimulation involves the simultaneous delivery of two pure sound waves of different frequencies to each ear, with the brain perceiving the mathematical difference between these two frequencies as a third, internally generated beat. In this protocol, a carrier frequency of 120 Hz will be delivered to the right ear and 100 Hz to the left ear, generating a binaural difference of 20 Hz corresponding to the Beta frequency band (~20 Hz), which is associated with cognitive alertness and motor focus. Carrier frequencies in the 100-120 Hz range were selected as they are physiologically safe and well-tolerated by the human ear (Chaieb et al., 2015).

   The binaural audio file was created using Audacity (version 3.7.5.0) software, with the two frequency channels carefully separated to prevent mixing. Pink noise was added to the background of the binaural stimulus to enhance listening comfort and mask environmental sounds without physically occluding the ear canal.

   Since the literature indicates that binaural stimulation requires a neurological adaptation period to fully demonstrate its targeted cortical effects (Gao et al., 2014), participants will listen to the binaural audio file for 6 minutes in a resting position immediately before commencing the main walking test (Ala et al., 2018). The binaural stimulus will then continue to be delivered throughout the 6MWT via open-ear headphones.

3. Control Condition Participants will complete the 6MWT without wearing headphones and without any additional auditory stimulus, under standard clinical environmental sound conditions.

Equipment Headphones: Auditory stimuli will be delivered via HUAWEI FreeClip 2 headphones. These headphones employ next-generation Open-Ear (Directional Air-Conduction) technology. The acoustic element sits just outside the ear canal rather than inside it, directing sound into the canal while leaving the ear canal physically unoccluded. This design ensures that participants can fully perceive standardized verbal motivational commands from the researcher and ambient environmental sounds throughout the test, without compromising the integrity of auditory stimulus delivery.

Pulse Oximeter: Heart rate and peripheral oxygen saturation (SpO₂) will be measured using a fingertip pulse oximeter (Surginatal, Lk47, China).

Sphygmomanometer: Blood pressure will be measured using an automated sphygmomanometer (Omron, HEM-7143-E, Netherlands).

Study Procedures Pre-Test Assessment At the beginning of each session, participants will rest for 10 minutes in a seated position. Following this rest period, baseline measurements will be recorded: heart rate, blood pressure, SpO₂, and perceived dyspnea, general fatigue, and quadriceps femoris muscle fatigue using the Borg Scale (6-20). Participants will be asked to rate each of these three perceptual dimensions separately on a scale from 6 (no exertion at all) to 20 (maximal exertion).

6-Minute Walk Test (6MWT) The 6MWT will be conducted in accordance with ATS/ERS standards (ATS Committee, 2002) in a straight, flat, marked corridor of 30 meters in length, with emergency intervention equipment accessible. Participants will be instructed to walk as far as possible at their own pace without running for 6 minutes. Standardized verbal encouragement will be provided at each minute mark. If needed, the participant will be allowed to rest without the timer being stopped. For participant safety, the test will be immediately terminated by the researcher in the event of chest pain, severe dyspnea, loss of balance, leg cramps, or excessive pallor/diaphoresis.

At the end of the 6-minute period, the total distance covered will be recorded in meters. Walking cadence will be assessed as an additional measure of gait dynamics and effort efficiency: during the 4th minute of the 6MWT, the researcher will count and record the number of steps taken by the participant over 1 minute (Deboeck et al., 2005).

Post-Test Recovery Period Immediately following test completion and at the 1st, 3rd, and 5th minutes of the recovery period, the following measurements will be reassessed and recorded: heart rate, SpO₂, blood pressure (in seated position), and Borg Scale scores for perceived dyspnea, general fatigue, and quadriceps femoris muscle fatigue.

Measurement Time Points Summary

All physiological and perceptual parameters will be recorded at five time points per session:

Pre-test (resting baseline) Immediately post-test Recovery minute 1 Recovery minute 3 Recovery minute 5

Sample Size The minimum sample size required to ensure adequate statistical power will be determined following a pilot study conducted at the beginning of the data collection process. The actual effect size specific to this study will be calculated from the pilot data and entered into G*Power software. The net number of participants required for a significance level of α = 0.05 and a statistical power of 80% will be objectively determined, and a 20% buffer will be added to account for potential data loss.

Statistical Analysis All statistical analyses will be performed using SPSS version 26. Descriptive statistics will be expressed as mean ± standard deviation (M ± SD) for continuous variables and as frequency and percentage (n, %) for categorical variables. The normality of data distributions will be assessed using both visual methods (histograms and Q-Q plots) and analytical methods (Shapiro-Wilk test).

Given the cross-over design of the study, One-Way Repeated Measures ANOVA will be used to examine the effect of the three auditory conditions (binaural beats, metronome, control) on 6-Minute Walk Distance (6MWD) and mean walking cadence. To control for potential learning or fatigue effects arising from the order in which participants underwent the test conditions, the participant's sequence assignment will be included as a between-subjects factor in the analysis.

Two-Way Repeated Measures ANOVA (condition × time) will be applied to examine the main effects of the three auditory conditions and the condition × time interaction on autonomic and physiological parameters across the five measurement time points (pre-test, post-test, recovery minutes 1, 3, and 5).

Since Borg Scale scores for dyspnea and fatigue represent ordinal data, the non-parametric Friedman Test will be used directly for these variables. Where statistically significant differences or interactions are identified, Bonferroni-corrected post-hoc tests will be applied to determine which conditions or time points are responsible for the observed differences. The significance level for all statistical analyses will be set at p < 0.05.

Safety Measures All test procedures will be conducted by experienced physiotherapists, and participants will be under continuous physiotherapist supervision throughout the test. Vital signs will be assessed prior to each test session to exclude contraindicated conditions. The test will be immediately terminated by the physiotherapist if any of the following occur during the session: chest pain, intolerable dyspnea, loss of balance, leg cramps, excessive pallor or diaphoresis, or sudden onset headache, nausea, or vertigo potentially related to binaural stimulation.

Study Duration: April - June 2026 (3 months)

Inclusion Criteria:

• Aged between 18 and 35,
• Able to walk unaided (without the use of a walking aid),
• Participants who agree to take part in the study on a voluntary basis and sign the informed consent form

Exclusion Criteria:

• A history of myocardial infarction or unstable angina within the last month,
• Acute illness or pain during the test that would prevent walking,
• A lower limb injury or surgery within the last 3 months,
• Resting systolic blood pressure >180 mmHg or diastolic blood pressure >100 mmHg,
• Resting heart rate exceeding 120 beats per minute,
• Those with cardiovascular or pulmonary conditions that may limit exercise,
• Those with neurological conditions (balance disorders, stroke, epilepsy, etc.),
• Those with musculoskeletal problems (lower limb fracture, severe arthritis, acute injury, etc.),
• Those with an acute infection, fever or severe fatigue that could prevent walking during the test,
• Those with a body mass index of 35 or above (obesity),
• Those with a history of active vertigo, Meniere's disease or vestibular system disorders,
• Participants with a diagnosed hearing problem

The study population consists of healthy young adults aged 18-35 living or studying in Gaziantep, Turkey. Participants will be recruited from students and staff at Hasan Kalyoncu University who meet the inclusion criteria. All participants must be able to walk independently without an assistive device and must provide signed informed consent. Individuals with cardiovascular, pulmonary, neurological, or musculoskeletal conditions, active vestibular disorders, diagnosed hearing problems, resting blood pressure above 180/100 mmHg, resting heart rate above 120 bpm, or a BMI of 35 or above will be excluded. This young adult population was specifically selected to eliminate age-related confounders

The data collected will be shared by the principal investigator with the relevant individuals, without including patients' names.