Breathwork and Stress: Investigating the Mechanisms of Action and Effectiveness of Breathing Interventions in Modulating the Psychophysiological Response to Acute Stress Test

Overview

This study investigates whether the psychophysiological benefits of breathing exercises are driven by a specific physiological rhythm (6 breaths per minute) or by the general psychological experience of performing a structured, mindful activity. Researchers aim to determine if "coherent breathing", which is hypothesized to synchronize heart and respiratory rhythms, offers unique physiological protection against stress compared to breathing at a natural pace or simple resting. The main questions it aims to answer are: * Does slow, steady breathing at 6 breaths per minute lower physical stress markers (like heart rate variability and cortisol) better than faster, but structured breathing or just sitting still? * Is the calming effect caused by the specific breathing rhythm or simply by performing a structured, relaxing activity? Researchers will compare three groups to see if the specific rhythm of "coherent breathing" offers unique benefits: 1. Group (Interventional): Coherent Breathing: Slow breathing at 6 breaths per minute. 2. Group (Sham Breathing): Regular breathing at 15 breaths per minute (matching a natural pace). 3. Spontaneous Breathing (Control Group): Natural, unguided breathing. Participants will: * Complete a one-day preparation phase to become familiar with the breathing technique. * Visit the research center for one experimental session. * Perform their assigned breathing method before and after a stress test. * Take the Maastricht Acute Stress Test (MAST), which involves putting a hand in cold water and doing mental math. * Provide saliva samples and have their heart rate variability, and mood measured multiple times.

Detailed description

This three-arm randomized controlled trial is designed to investigate whether the therapeutic benefits of slow-paced breathing stem from specific physiological mechanisms (cardiorespiratory resonance) or non-specific psychological factors, such as attention, expectancy, and structured relaxation.

The study directly compares resonant-frequency breathing against a structurally identical sham intervention and a passive control group.

Experimental Protocol:

The study utilizes the Maastricht Acute Stress Test (MAST) as a highly structured and validated laboratory stressor to capture the dynamic psychophysiological response to acute distress.

The protocol consists of the following phases:

Baseline Phase (BASE): A 5-minute measurement of baseline physiological (HRV - Heart Rate Variability) and psychological (STAI-S - State-Trait Anxiety Inventory-State, VAMS - Visual Analogue Mood Scales) parameters.

First Intervention (10 min): Participants perform their assigned breathing technique or sit quietly to assess the preventive "buffering" effect of the intervention.

Acute Stress Induction (MAST): A 15-minute structured stressor combining periods of hand immersion in cold water and mental arithmetic under social-evaluative pressure. The water temperature is strictly maintained within the range of 4.5 - 5.5 °C using a TECO chiller to ensure consistent stress induction across all participants.

Post-Stress Recovery (POST): Immediate 5-minute measurement following the conclusion of the MAST protocol.

Second Intervention (10 min): A repeat of the breathing/control protocol to evaluate its impact on psychophysiological recovery.

Final Assessments (END \& FOLLOW): Measurements are taken immediately after the second intervention and following a 15-minute recovery phase to capture the peak glucocorticoid (cortisol) response.

Data Collection and Analysis:

* Heart Rate Variability: RR interval (time duration between successive heartbeats, specifically the time intervals between each R-wave peak) data is collected using a Polar H10 heart rate monitor. To ensure data integrity and redundancy, the signal is recorded simultaneously through two mobile applications: Elite HRV and Kubios HRV. All subsequent advanced HRV analyses, including time-domain, frequency-domain, and non-linear indices (e.g., PNS/SNS Index \[Parasympathetic Nervous System Index and Sympathetic Nervous System Index\], RMSSD \[Root Mean Square of Successive Differences\]), are performed using Kubios HRV Scientific software. * Biochemical Markers: Salivary cortisol levels are measured to assess the hypothalamic-pituitary-adrenal (HPA) axis response using ELISA kits. * Psychological Scales: Subjective stress and anxiety are tracked using the STAI-VAMS.

Adherence Check: Participant adherence to the paced breathing protocols is verified using the Kubios RESP (respiratory rate estimation) algorithm. This software-based solution extracts respiratory frequency from the RR interval data to confirm that participants maintained the target rates of 6 bpm (breaths per minute) or 15 bpm.

Research Arms:

1. Coherent Breathing (6 bpm): Guided nasal diaphragmatic breathing (5.5 s inhale / 5.5 s exhale) focused on inducing cardiorespiratory resonance. 2. Sham Breathing (15 bpm): Guided breathing at a natural pace (2 s inhale / 2 s exhale) to control for the effects of a structured intervention. 3. Spontaneous Breathing (Control): Passive resting with eyes closed, serving as a neutral baseline for the natural course of the stress response.

Interventions

• Behavioral Coherent Breathing (6 bpm)
  A 10-minute session of guided nasal diaphragmatic, coherent breathing. Participants follow rhythmic audio bell cues: a low tone for a 5.5-second inhalation and a high tone for a 5.5-second exhalation. Instructions emphasize silent, gentle nasal breathing and abdominal (diaphragmatic) expansion without lifting the shoulders. Participants are asked to focus their attention on the sensations of air flow or abdominal movement throughout the session. The intervention is performed twice: before and af
• Behavioral Sham Breathing (15 bpm)
  A 10-minute session of guided nasal diaphragmatic breathing at a rate of 15 breaths per minute (matching a natural spontaneous pace). Participants follow identical rhythmic audio bell cues as the experimental group: a low tone for a 2.0-second inhalation and a high tone for a 2.0-second exhalation. All other instructions regarding nasal breathing, abdominal mechanics, and mindful attention are identical to the experimental group to ensure structural blinding. The intervention is performed twice:

Primary outcome measures

• Change in Root Mean Square of Successive (RMSSD) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in PNS Index [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in Salivary Cortisol Concentration [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in STAI-State score [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in Mean HR [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]

Secondary outcome measures (12)

• Change in SNS Index [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in Diastolic Blood Pressure (DBP) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in Subjective Stress (VAMS) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Mean Heart Rate (Mean HR) during Experimental Blocks [Time frame: Average heart rate measured during three 10-minute periods starting at approximately 10 minutes (First Intervention), 35 minutes (Stress Induction), and 55 minutes after Baseline during the single experimental Day 3.]
• SNS Index during Stress Induction [Time frame: Average score measured during one 10-minute period starting at approximately 35 minutes (Stress Induction) after Baseline during the single experimental Day 3.]
• Change in Standard Deviation of NN intervals (SDNN) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• SNS index during Intervention Blocks [Time frame: Average score measured during two 10-minute periods starting at approximately 10 minutes (First Intervention) and 55 minutes (Second Intervention) after Baseline during the single experimental Day 3.]
• Change in LF power log (ln LF) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• Change in Log-transformed High-Frequency HRV Power (ln HF) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]
• PNS Index during Stress Induction [Time frame: Average score measured during one 10-minute period starting at approximately 35 minutes (Stress Induction) after Baseline during the single experimental Day 3.]
• PNS index during Intervention Blocks [Time frame: Average score measured during two 10-minute periods starting at approximately 10 minutes (First Intervention) and 55 minutes (Second Intervention) after Baseline during the single experimental Day 3.]
• Change in Systolic Blood Pressure (SBP) [Time frame: Measured at 5 time points: at Baseline (0 minutes), and approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.]

Eligibility criteria

Inclusion criteria

• Healthy adults
• Aged 18 to 60 years
• Willingness to participate in all study phases, including preparation and laboratory session.
• Professionally active individuals or university students.

Exclusion criteria

• Severe chronic diseases, including metabolic disorders (e.g., diabetes) and mental disorders.
• Cardiac arrhythmia, history of heart attacks, strokes, or heart surgery.
• Regular use of medications such as anxiolytics or beta-blockers (excluding hormonal contraception)
• Pregnancy.
• Current participation in other scientific experiments.
• Significant previous experience with breathing techniques or current independent breathwork/meditation practice (defined as regular practice for more than 7 days in total within the last 12 months).
• Professional sports practice.
• Raynaud's disease
• Inability to abstain from alcohol, caffeine, and nicotine for the required periods before the experiment

Criteria are shown verbatim from the registry (in English). Final eligibility is always assessed by the study center.

Healthy volunteers: Yes

Study design

Allocation

Randomized

Model

Parallel assignment

Masking

Single blind

Primary purpose

Basic science

Study locations

Poland · 1 center

• Medical University of Bialystok. Department of Physiology. — Bialystok

Publications

• Dick TE, Hsieh YH, Dhingra RR, Baekey DM, Galan RF, Wehrwein E, Morris KF. Cardiorespiratory coupling: common rhythms in cardiac, sympathetic, and respiratory activities. Prog Brain Res. 2014;209:191-205. doi: 10.1016/B978-0-444-63274-6.00010-2. PMID 24746049
• Sevoz-Couche C, Laborde S. Heart rate variability and slow-paced breathing:when coherence meets resonance. Neurosci Biobehav Rev. 2022 Apr;135:104576. doi: 10.1016/j.neubiorev.2022.104576. Epub 2022 Feb 12. PMID 35167847
• Siebieszuk A, Plonski AF, Baranowski M. Breathwork for Chronic Stress and Mental Health: Does Choosing a Specific Technique Matter? Med Sci (Basel). 2025 Aug 13;13(3):127. doi: 10.3390/medsci13030127. PMID 40843749
• Smeets T, Cornelisse S, Quaedflieg CW, Meyer T, Jelicic M, Merckelbach H. Introducing the Maastricht Acute Stress Test (MAST): a quick and non-invasive approach to elicit robust autonomic and glucocorticoid stress responses. Psychoneuroendocrinology. 2012 Dec;37(12):1998-2008. doi: 10.1016/j.psyneuen.2012.04.012. Epub 2012 May 18. PMID 22608857
• Shilton AL, Laycock R, Crewther SG. The Maastricht Acute Stress Test (MAST): Physiological and Subjective Responses in Anticipation, and Post-stress. Front Psychol. 2017 Apr 19;8:567. doi: 10.3389/fpsyg.2017.00567. eCollection 2017. PMID 28469586

Identifiers

NCT: NCT07529379 · APK.002.107.2024

Primary sources (government registries)

• ClinicalTrials.gov (US NIH)

View this study on ClinicalTrials.gov ↗