Effectiveness of music with auditory beat stimulation in reducing state anxiety in Canadian students with trait anxiety: protocol for a randomised controlled trial

Abstract

Introduction

Undergraduate students report a high level of trait anxiety, which is a risk factor for further psychological decline if unmanaged. Music-based interventions are cost-effective and have been found to improve indices of anxiety. More recently, music with auditory beat stimulation (ABS) has been shown to improve symptoms of anxiety to a greater extent than music alone. While there is limited empirical evidence, music interventions with ABS may also be effective at targeting neurophysiological markers of anxiety. The aim of this study is to evaluate the effectiveness of a novel music with ABS intervention on self-report and neurophysiological indices of anxiety in undergraduate students with trait anxiety. It is hypothesised that relative to a pink noise control, listening to music with ABS will lower self-reported anxiety, reduce salivary cortisol, increase heart rate variability, increase theta and alpha-band electroencephalography (EEG) power and decrease beta and gamma-band EEG power.

Methods and analysis

Fifty Canadian undergraduate students who self-report experiencing anxiety will be recruited for this two-arm randomised controlled trial. Participants will be randomised to a single music session with ABS or pink noise; each intervention ranges from 24 min to 27 min. Outcomes will be assessed at baseline and immediately following the intervention and will be self-reported anxiety and affect (the State-Trait Inventory of Cognitive and Somatic Anxiety and the Self-Assessment Manikin), salivary cortisol, heart rate variability measured by ECG and cortical measures of anxiety (measured by EEG). Repeated measures analyses of covariance will be performed to evaluate the effect of condition assignment on outcome measures.

Ethics and dissemination

This study will be conducted under the Declaration of Helsinki. This study was approved by the Toronto Metropolitan University Research Ethics Board (REB-2020-068) and registered on ClinicalTrials.gov (NCT05442086). The findings of this study will be published in a peer-reviewed journal.

Trial registration number

NCT05442086.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• This study employs multiple measures (self-report, salivary cortisol, heart rate variability and spectral power analysis of electroencephalogram) to index anxiety in response to auditory interventions.

• This study adds to a sparse body of literature on neurophysiological mechanisms of action for anxiety reduction following auditory interventions.

• The study will be conducted on undergraduate students. While this may be considered a strength, given high rates of anxiety in students, it may also be considered a constraint on the generality of findings

Background

In 2022, 36% of young adults in the USA reported experiencing anxiety¹; a stark increase from 15% in 2018.² This may be driven by the consequences of the COVID-19 pandemic, such as strain on familial relationships³ or social anxiety following isolation,⁴ which compound the typical anxieties young adults experience.⁵ Anxiety is associated with high healthcare costs,⁶ and anxiety disorders are a leading cause of disability.⁷ Often, the burden of treatment costs falls on the individual. In 2006, it was estimated that the total cost per person for managing an anxiety disorder in the USA was US$6475,⁸ making it challenging to access care⁶ and leaving many undertreated.⁹ This is of vital concern, as unmanaged anxiety can lead to further decline in mental health¹⁰ and a high allostatic load that carries an increased risk for later cardiovascular and metabolic illness.¹¹

Individuals at risk of developing an anxiety disorder tend to have higher levels of trait anxiety.¹² Trait anxiety is a characteristic marked by proneness to perceiving threats, becoming anxious in a given situation or experiencing chronic mismatches between environment and expectations.^{13 14} Ree et al¹³ conceptualised trait anxiety as consisting of cognitive (e.g., worry, lack of concentration and intrusive thoughts) and somatic (e.g., trembling, hyperventilation and sweating) responses to perceived stressors. In other words, trait anxiety increases one’s susceptibility to experiencing high-state anxiety in a given circumstance. This exaggerated stress response is associated with a heightened sympathetic nervous system activity. This is evidenced by findings that trait anxiety is associated with decreased heart rate variability (HRV), theorised to arise from a reduced ability of the parasympathetic nervous system to regulate the sympathetic nervous system.15,17

Additionally, the hypothalamic-pituitary-adrenal (HPA) axis, the second primary stress system, may be amplified in individuals with moderate to high trait anxiety, denoted by an elevated cortisol response to stressful situations¹⁸ and higher awakening cortisol response,¹⁹ relative to those without anxiety. Both elevated cortisol and decreased HRV can be risk factors and biomarkers of a psychiatric disorder, such as anxiety and depressive disorders.^{20 21}

Developing interventions that are cost-effective and that target psychological and physiological indices of anxiety is critical. The gold-standard non-pharmacological intervention for anxiety is cognitive–behavioural therapy (CBT; see Stewart and Chambless²² and Hoffman and Smits²³ for reviews). However, one of the challenges of psychotherapy is that personal factors, such as patient resistance or ambivalence towards treatment, are a central predictor of its effectiveness.²⁴ While there are well-validated treatments targeting resistance,²⁵ attitudinal factors can be challenging to overcome, leaving limited treatment options for those who are not ready for psychotherapy. A second factor that may pose a barrier to CBT is cost. While increased accessibility of CBT through cost reduction is viable with the emergence of e-health, a 16-week course of CBT can still range at a minimum of US$778–US$957.²⁶ Lastly, the severity of an anxiety disorder is a strong predictor of seeking treatment.²⁷ As such, individuals with trait anxiety who do not meet the criteria for an anxiety disorder may be more reluctant to seek treatment. Nevertheless, these populations could still benefit from anxiety management.

Given that music listening tends to be widely practised and the most common leisure activity used for mood management,²⁸ music-based interventions may offer a dynamic tool that leads to greater engagement and mental health treatment for individuals who experience barriers to accessing standard care.²⁹ Music listening has been used to increase tolerance of difficult emotions in both formal clinical interventions (eg, dialectical behavioural therapy³⁰) and informally, by the general population,^{28 31 32} suggesting broad acceptability across contexts. Furthermore, when implemented in pre-existing healthcare systems, music-based interventions are found to be highly cost-effective—reducing healthcare costs by 22%, relative to treatment as usual.³³

There are specific benefits of music-based interventions for anxiety reduction. One meta-analysis of 19 studies found that an average of 22 min of music listening sessions reduced self-reported anxiety in non-clinical samples.³⁴ Another meta-analysis found that randomised controlled trials (RCTs) using music therapy reduced anxiety outcomes across diverse cultures and population groups (eg, younger and older adults, non-clinical and clinical) immediately following an intervention.³⁵ In studies that conducted follow-up assessments (n=5), two studies, one with incarcerated adults³⁶ and the other with older adults with dementia,³⁷ found sustained effects on anxiety at one week and eight weeks post-intervention, respectively. However, given that only five studies conducted follow-up tests, the lasting effects of anxiety reduction following music interventions and the characteristics of the intervention and population that render interventions most effective are areas for future research.

An important biomarker of stress is cortisol. A systematic review found that the most common stress biomarkers studied in music intervention research are plasma cortisol, salivary cortisol and α-amylase.³⁸ The same review found a significant reduction in stress biomarkers, which was associated with reduced psychological stress. One study showed that individuals who listened to personal self-selected and neutral experimenter-selected music at home and in a laboratory environment significantly reduced cortisol levels. This was found regardless of the music type and setting, although baseline cortisol levels were higher in the lab than at home.³⁹ Importantly, music therapy is effective at anxiety reduction, while the evidence for the effects of psychophysiological measures of anxiety such as reduced cortisol and heart rate is mixed.³⁴ These mixed findings can be attributed to several exogenous sources such as individual variability, measurement techniques and, importantly, temporal differences in cortisol release due to fluctuations in circadian rhythm.⁴⁰

In addition to the psychophysiological measures, neurophysiological data provide additive support for the role of music in anxiety reduction. There are several ways to measure and describe anxiety in electroencephalography (EEG) research. One simple interpretation is that anxiety tends to be associated with increased power in higher frequency bands, such as beta waves (13–30 Hz), and decreased power in lower frequency bands, such as alpha waves (8–12 Hz) and theta waves (4–7 Hz). Increased beta wave power is purported to indicate heightened arousal and vigilance,⁴¹ which are markers associated with anxiety. Conversely, decreased alpha and theta wave power are purported to be linked to difficulties in relaxation and attentional control, which are common impairments associated with anxiety.⁴²

The experimental findings corroborate this theory. For instance, the most prominent signature pattern of anxiety is excessive beta power emanating from a source in the anterior cingulate cortex and, more generally, recorded over midline cortical regions.⁴³ One study showed that individuals who present high trait anxiety exhibit dysregulation in theta-band event-related synchronisation in frontal regions when viewing images of threatening stimuli.⁴⁴ On the other hand, a wide variety of interventions that aim to reduce anxiety, such as virtual reality and progressive muscle relaxation, tend to show a reduction in power in higher frequency bands, such as beta (13–30 Hz), and an increase in lower frequency bands, such as alpha (8–10 Hz) and theta (4–7 Hz).^{45 46} Given that various anxiety-reducing interventions consistently reveal an effect of heightened power in slow-wave oscillations, particularly in the theta band, this pattern should be generalised across novel interventions. This implies that a similar outcome for this effect can be replicated for music-based interventions.

More recently, other auditory techniques for reducing anxiety have been explored. Auditory beat stimulation (ABS), specifically binaural beats, has gained popularity for its potential anxiolytic effects,47,51 cognitive effects52,55 and, in some cases, improved psychophysiological states.^{56 57} Binaural beats are generated by placing tones of differing frequency in each ear. The difference between the frequencies will be experienced as a perceivable beat frequency; for example, 400 Hz in the right ear and 405 Hz in the left ear would be perceived as a beat, or amplitude modulation, based on the combined neural response at 5 Hz. Although much of the anxiety research assessing binaural beats has demonstrated the expected reduction, the effectiveness of ABS has not been consistently supported.58,63 This inconsistency is likely due to several factors which include specific frequency of stimulation, type of sound used to mask the binaural beat, trait anxiety, personality factors such as extraversion and the duration of exposure.4964,68 In prior work by the authors⁴⁸ and in the current study, these factors are accounted for through experimental design and statistics. Binaural beats with a frequency of stimulation between 4 and 7 Hz, considered to be in the theta frequency range, have consistently been demonstrated to evoke a relaxation response.69,71 Moreover, the effects of combined music and ABS on anxiety appear to be additive.⁴⁸ The mechanism by which ABS reduces anxiety has only recently begun to be explored. Low-frequency ABS appears to modulate neural activity via cortical and subcortical neural entrainment to the beat at the stimulated frequency.⁷² This entrainment of low-frequency neural oscillations may elicit downstream effects on the balance of autonomic nervous system (ANS) activity, leading to higher HRV.⁷³ To the best of our knowledge, this is one of the first clinical trials on the topic of anxiety, music and ABS that combines multiple objective psychophysiological measures (i.e., spectral power EEG, HRV and salivary cortisol) with subjective measures of mood and anxiety.

The purpose of the present trial is to replicate and expand on a subset of the findings of Mallik and Russo⁴⁸ by investigating the effectiveness of music with theta-band ABS for reducing state anxiety in individuals with trait anxiety. Four hypotheses were generated for this aim. First, participants who listen to music with ABS will report a greater reduction in self-reported state anxiety than those who listen to pink noise (control). Second, participants in the music with ABS condition will show reduced salivary cortisol levels relative to the pink noise condition. Third, it is expected that participants in the music with ABS condition will show increased HRV compared with the pink noise condition. Finally, frequency spectral power analysis of EEG from participants in the music with ABS condition will show increased theta and alpha-band power and decreased beta and gamma-band power compared with the EEG from participants in the pink noise condition.

A series of exploratory analyses will be conducted to elucidate potential neurophysiological mechanisms by which music with ABS reduces state anxiety and generate future hypotheses. In line with recent findings on the mechanisms of ABS,⁷² we will explore if music with ABS will entrain the cortex at the beat frequency to a greater extent than the pink noise control condition. Additionally, we will explore between-group differences in frequency band power. To this end, it is postulated that the benefits of music with ABS may arise from higher alpha and theta power in the alpha/beta and theta/beta frequency ratios, respectively74,76; frontocortical asymmetry in alpha power (i.e., greater left frontal activity as indicated by lower alpha power)^{77 78}; and lower gamma power.79,81 These cortical measures will be analysed independently and in conjunction with HRV measures associated with stress.¹⁵ To date, none of these EEG biomarkers have been tested empirically in the context of music and ABS. Therefore, analyses will be conducted to observe which, if any, differences in frequency band power exist between the music with ABS and pink noise conditions.

Methods

Overview

The study is a two-arm (treatment vs. control condition), parallel-design RCT. Following screening for eligibility and consent, participants will be randomised to the music with ABS condition (treatment) or the pink noise condition (control) at a rate of 1:1. Baseline measures will be assessed, including self-reported state affect, trait and state anxiety, electrophysiological recordings of cardiac (ECG) and neuroelectric (EEG) activity and salivary cortisol. Immediately following the baseline recordings, participants will be exposed to their assigned audio-based intervention: pink noise or music with ABS, while ECG and EEG measurements continue. Following the intervention, ECG and EEG measurements will continue for 1 min, and a second salivary cortisol sample will be collected. Finally, participants will complete measures of self-reported state anxiety and affect and will provide a third saliva sample.

Standard Protocol Items: Recommendations for Interventional Trials statement

This protocol was written in accordance with the 2013 Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) reporting guidelines.⁸² See online supplemental appendix A for the SPIRIT statement.

Patient and public involvement

There was no patient or public involvement in the design of this study protocol.

Study settings

The present study will be conducted at Toronto Metropolitan University (TMU), an urban university in Toronto, Ontario, Canada. Participants will be recruited from TMU’s undergraduate participant pool or the Toronto community and will complete the experiment on-site. Prior research has found high rates of trait anxiety in undergraduate students, with 60% having trait anxiety.⁸³

Eligibility criteria

The inclusion criteria are that participants must be comfortable speaking and understanding English, an undergraduate student, between the ages of 18 and 30, have self-reported normal hearing and have a score of at least 1 on the Generalized Anxiety Disorder-2 screening tool. This indicates feeling anxious or unable to control worry for several days within a 2-week period.⁸⁴ Exclusion criteria are cardiac issues and a history of seizures or epilepsy. Recruitment began on 10 August 2024. The first participant was enrolled on 16 August 2024, and the anticipated completion date is 1 September 2025.

Informed consent

Written informed consent will be obtained before the scheduled in-lab visits (see online supplemental appendix B for consent form). Participants will be emailed a link to sign an online consent form before their appointment and will be encouraged to contact the researchers with any questions. If participants are undecided about providing consent, they will be given the option to speak with a member of the research team prior to commencing any research activities. Participants will be allowed to cease participation at any time, at which point the experiment will end, and their data will be deleted.

Intervention

Participants will be informed that they are participating in a research study examining the effects of auditory interventions for students with anxiety. Participants will be randomly assigned (1:1) to one of two audio interventions: a single session of listening to approximately 25 min of personalised music with ABS in the theta frequency range (4–7 Hz; treatment condition) or a single session of 24 min of pink noise (control condition), both supplied by LUCID Therapeutics. The treatment condition employs LUCID’s proprietary algorithms for implementation of the iso principle⁸⁵ and integration of auditory beat stimuli into music ⁸⁶. A widely used approach in music therapy, the iso principle posits that to maximise the calming benefits of music, a piece of music should initially align with the listener’s current emotional state and gradually shift towards their desired emotional state.⁸⁷ The music playlist is personalised to each listener’s affective state, taking into account their baseline valence and arousal scores provided on a 5-point Self-Assessment Manikin (SAM).⁸⁸ The algorithm references these baseline scores against its own predictions about valence and arousal conveyed in each piece within a database of music. This method of self-reporting affect and indexing music affect is based on the circumplex model of affect, encompassing a range of emotions varying with respect to valence and arousal.⁸⁹ In Mallik and Russo,⁴⁸ the same personalised music with the ABS system was found to be more effective in reducing cognitive symptoms of anxiety in individuals with moderate trait anxiety relative to music alone, ABS alone and pink noise.

Pink noise was selected as a control condition, having been used previously in a previous foundational study conducted by two of the authors of the current study.⁴⁸ Many prior studies involving auditory stimuli have used pink noise as a control stimulus.^{90 91} Pink noise is similar to a silent control condition while controlling for sound stimulation.⁹² In addition, the advantage of pink noise as a control condition compared with the silent condition is that it helps control for a placebo effect. To this end, pink noise was administered in the same manner as all the other stimuli (i.e., there would be no indication that this was a control). Those in the pink noise control condition will also rate their valence and arousal before listening; however, this has no impact on the audio they are exposed to.

Participants will listen to their assigned audio intervention in a double-walled, sound-attenuated chamber (Industrial Acoustics, Bronx, New York). The audio will be presented through 3M insert earphones to avoid the potential for electromagnetic contamination of EEG recordings. The audio intervention will be presented on a Dell Latitude 3540 Laptop. Prior to the intervention, participants will listen to a brief clip of the audio intervention they are assigned to and will be allowed to adjust the volume to a comfortable listening level. For the duration of the intervention, they will be seated comfortably in a reclining chair in the sound-attenuated chamber with instructions to wear a blindfold or close their eyes/soften their gaze if they prefer.

Primary outcome measures

Cognitive and somatic state anxiety

The State-Trait Inventory for Cognitive and Somatic Anxiety (STICSA¹³) is a 21-item measure designed to measure somatic (11 items) and cognitive symptoms (10 items) of anxiety at both the trait and state levels. To differentiate between state and trait anxiety symptoms, participants will complete two versions of the measure, one where they rate how they ‘feel right now at this very moment’ (STICSA-State) and one where they rate ‘how they feel in general’ (STICSA-Trait). Respondents rate the degree to which each item is true for them on a Likert scale ranging from 1=not at all to 4=very much so. Scores on the STICSA can range from 10 to 40 (cognitive) or 11 to 44 (somatic), with higher scores indicating greater anxiety.

The STICSA-Trait will be included as a measure to assess trait anxiety, defined by cut-offs in Roberts et al.⁹³ The STICSA-Trait will be administered as part of a battery of pre-intervention questionnaires. The STICSA-State will be included in both the pre-intervention battery and following the intervention to assess changes from pre-intervention to post-intervention.

The STICSA demonstrates convergent validity with the State-Trait Anxiety Inventory (r=0.42–0.71) and the Depression, Anxiety and Stress Scale (r=0.42–0.61).⁹⁴ The STICSA also has strong internal consistency with α=0.87–0.92 for full scales and subscales (cognitive and somatic) of the STICSA-Trait and STICSA-State.⁹⁴

Affective valence and arousal

Two series of SAM⁸⁸ will be administered to assess state emotional valence and arousal before and after the audio intervention. Participants will be asked to rate on two scales with accompanying images how they are feeling right now (unhappy to happy) and their energy levels from calm to excited. The pre-intervention SAM serves two purposes: the first, to assess baseline affect, and the second, to select the playlist that the participant listens to if assigned to the audio intervention. The post-intervention SAM will serve to assess changes in affect following the intervention.

Salivary cortisol

Participants will provide saliva samples at three time points: 10 min before the intervention, one min after the intervention and 15 min after the intervention. Cortisol levels tend to peak between 10 and 30 min following stimuli⁹⁵; as such, we anticipate these measurements will capture HPA activity at baseline, during the intervention and post-intervention. All testing will occur between 12:00 and 19:00 to account for diurnal variation in cortisol.⁹⁵ Participants will be instructed to avoid consuming food, caffeine, nicotine, marijuana and vigorous exercise 120 min before their appointment. Participants will be asked before testing whether they complied with these instructions; any instructions that were not followed will be noted as potential confounds. A brief demographics questionnaire inquiring on sex, menstrual cycle, use of hormonal contraceptives and use of medications will be administered to participants to assess for any confounds.⁹⁶

At each time point, participants will be asked to provide 2 mL saliva in a cryovial supplied by Salimetrics. Vials will be placed in a refrigerator set to −80°C until they are ready to be assayed. Samples will be defrosted for 45 min before being assayed using the Salimetrics Gen 6 protocol.

Electrophysiological recording

EEG will be recorded using the BioSemi EEG system via 64 scalp electrode sites according to the international 10-20 electrode system as done in a previous study examining EEG response to meditation.⁷⁷ Electrodes placed on the mastoid process will serve as a reference. To capture artefacts arising from eye blinks, two external electrodes will be placed approximately one cm below and at the corner of each eye (four electrodes total). The BioSemi ActiveTwo EEG amplifier will be used to monitor scalp potentials and will be sampled at 2048 Hz. EEG will be stored as a continuous file, recording one min of baseline data, the duration of the audio intervention, and one min of post-intervention data, distinguished by manual triggers set by research assistants (RA). EEG data will be subject to an independent component analysis to identify and remove artefacts. Subsequently, band-pass filters will be applied to EEG data to remove noise and irrelevant frequencies. Average band power for theta (3.1–7.9 Hz), alpha (8–12 Hz), beta (12.1–24 Hz) and gamma (30–70 Hz) frequency bands⁹⁷ across the entire scalp for each time point (baseline and post-intervention) will be calculated.

ECG will also be recorded via the BioSemi system with two flat active electrodes attached to the participant’s left and right wrists. ECG will be measured for one min prior to the intervention, during the intervention and one min post-intervention. ECG samples will then be converted to measure HRV, as an index of ANS activity, using a time-based method (root mean square of the successive differences; RMSSD).

Secondary outcome measures

Anxiety Coping Measures Questionnaire

A questionnaire designed by the researchers will be administered to assess concomitant treatments or mechanisms participants use to manage anxiety symptoms (see online supplemental appendix C). Participants will be asked if they are taking any prescribed medication, complementary medicine (e.g., cannabinoid products) or practices (e.g., breathwork, meditation) to manage their anxiety symptoms. These will be reported as descriptive statistics and included in exploratory analyses or as covariates if there are significant between-group differences. This questionnaire will be administered in a battery of questionnaires before the intervention.

The Positive and Negative Affect Schedule

The Positive and Negative Affect Schedule (PANAS)⁹⁸ is a 20-item self-report questionnaire designed to measure two broad dimensions of affect: positive affect (PA) and negative affect (NA). Each dimension consists of 10 adjectives describing emotions (e.g., ‘excited’, ‘enthusiastic’ for PA; ‘distressed’, ‘upset’ for NA), and respondents rate the extent to which they have experienced each emotion over a specified time frame (e.g., ‘right now’, ‘past week’ or ‘in general’) using a 5-point Likert scale (1=very slightly or not at all, 5=extremely). The PANAS will be administered before and after the audio intervention to assess changes in affect. Scores on each dimension range from 10 to 50, with greater scores on the NA scale indicating greater NA (worse outcome) and greater scores on the PA scale indicating greater PA (better outcome).

The PANAS subscales demonstrate strong internal consistency with α=0.89 for the PA subscale and α=0.85 for the NA subscale.⁹⁸ The PANAS, when measuring momentary affect, demonstrates adequate external validity with the Hopkins Symptom Checklist (r=0.74 (negative subscale) and r=−0.29 (positive subscale)).

The Absorption in Music Scale

The Absorption in Music Scale (AIMS)⁹⁹ is a 34-item measure of individuals’ ability and willingness to allow music to draw them into an emotional experience. The AIMS was administered post-intervention, after completing all other post-intervention measures to avoid biasing responses. The AIMS will serve as a subgroup analysis to assess if individuals who report higher absorption respond better to the audio intervention in the treatment condition. The AIMS demonstrates strong convergent validity with the Tellegen Absorption Scale (r=0.76) and the Musical Involvement Scale (r=0.74).

Participant timeline

See Table 1 for an outline of the study schedule from enrolment to close out.

Table 1. Timeline of participation from enrolment to final measurement.

Time point	Enrolment	Start(Baseline)	Intervention	Post-Intervention	Post- Intervention T2
Enrolment
Eligibility screen (GAD-2)	X
Informed consent	X
Randomisation	X
Allocation	X
Interventions
Intervention group: 24–27 min of music with ABS			X
Control group: 24 min of pink noise			X
Assessments
STICSA-Trait		X
STICSA-State		X		X
SAM		X		X
PANAS		X		X
Anxiety coping measures		X
AIMS				X
Salivary cortisol		X		X	X
EEG		X	X	X
ECG		X	X	X

ABS, auditory beat stimulation; AIMS, Absorption in Music Scale; ECG, electrocardiogram; EEG, electroencephalography; GAD-2, Generalized Anxiety Disorder; PANAS, Positive and Negative Affect Schedule; SAM, Self-Assessment Manikin; STICSA, State-Trait Inventory for Cognitive and Somatic Anxiety.

Sample size

Fifty participants will be recruited to participate in this study. The sample size was determined based on an a priori power analysis from a previous study,⁴⁸ which suggests that to achieve a significant effect (p=0.05) at an appropriate statistical power (0.80) with an effect size (Cohen’s d) of 0.83, a minimum total sample size of 50 participants would be required.

Allocation and blinding

Randomisation will be determined once participants are enrolled in the study. Participants will be randomly assigned (1:1) to one of two interventions: the pink noise control condition or the music with ABS intervention. Random assignment will be done using an online randomiser tool (randomizer.org) controlled by a research coordinator who will not interact with participants. Participants will be informed that they will be listening to an unspecified audio intervention and will not be made aware that there are two conditions until they are debriefed following the completion of the study.

This study involves blinding. Two RAs will administer the study protocol and will be blind to condition assignment until the participant has been fitted with an EEG cap and seated in a sound-attenuated chamber. One RA (RA 1) will interact with the participant (e.g., administering questionnaires and collecting saliva samples) inside the sound-attenuated chamber for the duration of the experiment. A second RA (RA 2) will monitor ECG/EEG recordings, administer the audio intervention and have minimal interaction with the participant until the experiment is completed. Since RA 2 will administer the intervention, they will be aware of condition assignment, while RA 1 remains blinded throughout the study to avoid biased interactions with participants.

Data management and statistical analysis

Surveys will be administered via Qualtrics and all data will be stored per the institution’s data collection policy.

All outcomes (self-reported anxiety, cortisol, HRV and spectral power EEG) will be subject to repeated measures analyses of covariance (ANCOVA) models, with STICSA-Trait and AIMS scores entered as covariates. If the data do not satisfy the assumptions for the ANCOVA, permutation tests, specifically the permuco package in R, will be used to conduct a permutation version of the repeated measures ANCOVA.¹⁰⁰ Permutation tests can control the type I error rate for multiple comparisons; they are non-parametric and have no assumptions about the underlying distribution of the data that are common in other inferential statistical tests.101,103 Missing data will be addressed through listwise deletion, applied on a case-by-case basis for each analysis.

Four separate 2 (time) × 2 (condition) ANCOVAs will be employed to compare pre-post changes in STICSA-State, PANAS, affective valence and arousal between the two conditions (pink noise vs. music with ABS). The results will be reported, including the main effects of the experimental condition and time, and the interaction effects between the experimental condition and time. It is expected that those in the music with ABS condition will show a greater reduction on the STICSA-State, NA schedule and arousal measure, relative to the pink noise condition.

A 3 (time) × 2 (condition) ANCOVA will be employed to assess between-condition (pink noise vs. music with ABS) differences in salivary cortisol pre-intervention, during intervention and post-intervention. The results will be reported with the main effects of the experimental condition and time and interaction effects between the experimental condition and time. Tukey’s Honestly Significant Difference tests will be performed to assess any significant differences between means, correcting for multiple comparisons. Consistent with our hypothesis, it is predicted that those in the music with ABS condition will have lower salivary cortisol during the intervention, which will further decrease at post-intervention, relative to the pink noise condition.

A 2 (time) × 2 (condition) ANCOVA will be employed to assess between-condition (pink noise vs. music with ABS) differences in HRV pre-intervention and post-intervention. The results will be reported with the main effects of the experimental condition and time and interaction effects between the experimental condition and time. It is expected that those in the music with ABS condition will have lower HRV (converted to RMSSD) following the intervention, relative to the pink noise condition.

A 2 (time) × 2 (condition) × 4 (alpha, beta, theta and gamma bands) ANCOVA will be employed to assess between-group differences in EEG band power. The results will be reported with the main effects of the experimental condition and time and interaction effects between the experimental condition and time. If the model is significant, post hoc tests will be performed to further assess significant differences between means. It is expected that those in the music with ABS condition will show increased theta and alpha power and decreased beta and gamma power post-intervention, relative to those in the pink noise condition.

Monitoring

RAs will be present during all data collection activities, continuously monitor adherence and participant safety and upload participant data. There are no anticipated harms associated with participating in this research study. However, any adverse events will be reported to the TMU Research Ethics Board (REB).

Ethics and dissemination

This study involves human participants, and ethics approval was obtained from the TMU REB under the title: The effect of an affective music recommendation system and ABS on anxiety (approval number: REB-2020-068). The study was pre-registered on ClinicalTrials.gov (Identifier: NCT05442086). Changes to the protocol will be uploaded as amendments to the ClinicalTrials.gov registration and to the REB. Following the completion of the study, the findings will be disseminated through peer-reviewed journal publications and scientific presentations. The deidentified data from the study will be shared on an open repository, such as the Open Science Framework.