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

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.

Study Overview

• Status: Recruiting
• Conditions: Healthy; Healthy Adult Participants; Acute Stress Reaction; Stress Biomarkers
• Intervention / Treatment: Behavioral: Coherent Breathing (6 bpm); Behavioral: Sham Breathing (15 bpm)

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.

• Study Type: Interventional
• Enrollment (Estimated): 120
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Adam Siebieszuk, Master; Phone Number: +48 85 748 55 85; Email: adam.siebieszuk@sd.umb.edu.pl

Study Locations

• Poland
  • Podlaskie Voivodeship
    • Bialystok, Podlaskie Voivodeship, Poland, 15-222
      • Recruiting
      • Medical University of Bialystok. Department of Physiology.
      • Contact: Department of Physiology, Medical University of Bialystok; Phone Number: +48 (85) 748 55 85; Email: fizjolog@umb.edu.pl
      • Principal Investigator: Adam Siebieszuk, Master
      • Sub-Investigator: Marcin Baranowski, PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Coherent Breathing (6 bpm); Participants perform 10 minutes of guided nasal diaphragmatic breathing at a rate of 6 breaths per minute (~0.1 Hz). The practice involves a 5.5-second inhalation and 5.5-second exhalation phase, guided by rhythmic audio bell cues (low tone for inhale, high tone for exhale). Participants focus on abdominal movement and sensations of air flow. This intervention is performed twice: once before the acute stressor (MAST) and once during the recovery phase.	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 after the stress protocol; Other Names: Resonant Breathing
Sham Comparator: Sham Breathing (15 bpm); Participants perform 10 minutes of guided nasal diaphragmatic breathing at a rate of 15 breaths per minute, which matches a natural spontaneous breathing pace. The practice involves a 2.0-second inhalation and 2.0-second exhalation phase, guided by identical rhythmic audio bell cues as the experimental arm. This arm mimics the structural and attentional components of the active intervention without inducing cardiorespiratory resonance. Performed twice: before and after the MAST protocol.	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: before and after the stress protocol; Other Names: Normal-paced Breathing
No Intervention: Spontaneous Breathing (Control); Participants in this passive control group sit quietly with their eyes closed for 10 minutes. They receive initial brief relaxation instructions (two deep breaths with a sigh, body muscles relaxation) but do not receive any specific rhythmic guidance, audio cues, or instructions to modulate their breathing pattern. This arm serves as a baseline to control for the natural course of the stress response and the passage of time.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in Root Mean Square of Successive (RMSSD)	The Root Mean Square of Successive Differences (RMSSD) measured in milliseconds (ms). This is a time-domain HRV parameter used to assess parasympathetic nervous system activity. An increase indicates higher parasympathetic activity, while a decrease indicates a stress response.	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	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated modulation of vagal activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean RR interval, RMSSD, and the non-linear SD1 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoints for these measures. A value of 0 represents the population average; positive values indicate enhanced parasympathetic tone and recovery capacity.	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	Concentration Measured via enzyme-linked immunosorbent assay (ELISA) in nmol/L. This neuroendocrine marker assesses the activation of the hypothalamic-pituitary-adrenal (HPA) axis. It is used to quantify the objective impact of the breathing intervention on the peak hormonal stress response (typically reaching maximum concentration 15-30 minutes after the stressor).	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	A validated self-report questionnaire used to measure the current intensity of anxiety, tension, and nervousness. Scores range from 20 to 80, with higher scores indicating greater subjective psychological distress.	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	Mean heart rate measured in beats per minute (bpm). This serves as a fundamental indicator of physiological arousal and autonomic nervous system activity.	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

Outcome Measure	Measure Description	Time Frame
Change in SNS Index	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated physiological arousal and sympathetic activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean heart rate (HR), Baevsky's Stress Index, and the non-linear SD2 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoints for these measures. Positive values indicate sympathetic dominance and physiological arousal, providing a quantitative measure of the "cost" of the stressor and the effectiveness of the intervention in reducing sympathetic overdrive.	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)	Measured in millimeters of mercury (mmHg) using an automated sphygmomanometer. This metric serves as functional hemodynamic endpoint to verify the hypothesis that coherent breathing enhances baroreflex sensitivity and stabilizes blood pressure.	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)	A specific assessment of subjective stress intensity. Participants mark their current feeling on a 100 mm visual analogue scale (0-100), where 0 indicates "no stress" and 100 indicates "maximum stress" . Only this individual scale value is reported as the endpoint for this measure.	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	Continuous monitoring of mean heart rate (bpm) during active interventions and stress induction to evaluate real-time physiological reactivity and recovery dynamics.	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	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated physiological arousal and sympathetic activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean heart rate (HR), Baevsky's Stress Index, and the non-linear SD2 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoint for this measure.	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)	A time-domain metric reflecting the total variability of heart rate rhythm, measured in milliseconds (ms). It represents the overall capacity of the autonomic nervous system to adapt to internal and external stimuli.	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	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated physiological arousal and sympathetic activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean heart rate (HR), Baevsky's Stress Index, and the non-linear SD2 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoint for these measures.	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)	The natural logarithm of the HRV absolute power in the low-frequency band (0.04 - 0.15 Hz).	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)	The natural logarithm of the HRV absolute power in the high-frequency band (0.15 - 0.40 Hz).	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	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated modulation of vagal activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean RR interval, RMSSD, and the non-linear SD1 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoint for this measure.	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	This outcome measure is reported as a single composite score (standardized Z-score) representing the aggregated modulation of vagal activity. The final value is automatically calculated by Kubios HRV Scientific software from the mathematical combination of three specific assessments: mean RR interval, RMSSD, and the non-linear SD1 parameter, normalized against a reference database. Only the final aggregated Z-score will be reported as the endpoint for these measures.	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)	Measured in millimeters of mercury (mmHg) using an automated sphygmomanometer. This metric serves as functional hemodynamic endpoint to verify the hypothesis that coherent breathing enhances baroreflex sensitivity and stabilizes blood pressure.	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 Mental Tension (VAMS)	A specific assessment of subjective mental tension intensity. Participants mark their current feeling on a 100 mm visual analogue scale (0-100), where 0 indicates "no tension" and 100 indicates "maximum tension" . Only this individual scale value is reported as the endpoint for this measure.	At Baseline (0 minutes), and at approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.
Change in Subjective Calmness (VAMS)	A specific assessment of subjective calmness intensity. Participants mark their current feeling on a 100 mm visual analogue scale (0-100), where 0 indicates "no calm" and 100 indicates "maximum calm" . Only this individual scale value is reported as the endpoint for this measure.	At Baseline (0 minutes), and at approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.
Change in Subjective Relaxation (VAMS)	A specific assessment of subjective relaxation intensity. Participants mark their current feeling on a 100 mm visual analogue scale (0-100), where 0 indicates "no relaxation" and 100 indicates "maximum relaxation" . Only this individual scale value is reported as the endpoint for this measure.	At Baseline (0 minutes), and at approximately 25, 45, 60, and 75 minutes after Baseline during the single experimental session on Day 3.

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Depression Anxiety Stress Scales (DASS-21)	A 21-item self-report instrument used to characterize the participants' baseline psychological distress over the past week. Subscales for Depression, Anxiety, and Stress (scores 0-21 each) will serve as covariates to analyze the interaction between baseline state and intervention results.	At Baseline only (at the start of the Preparation Phase on Day 1).
State-Trait Anxiety Inventory (STAI-Trait)	Measures the participant's stable tendency to respond with anxiety. Scores range from 20 to 80. This trait measure will be used to analyze how personality-level anxiety influences the physiological response to the stressor and breathing tasks.	At Baseline only (at the start of the experimental session on Day 3).
Respiratory Rate (RESP)	Objective verification of respiratory frequency (breaths per minute) derived from RR interval data using the Kubios RESP algorithm. This measurement is used as a manipulation check to confirm participant adherence to the assigned breathing protocols (6 bpm for the experimental group and 15 bpm for the sham group) and to ensure the internal validity of the experimental blocks.	Average respiratory rate 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.

Collaborators and Investigators

• Sponsor: Medical University of Bialystok

Publications and helpful links

General Publications

• 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.
• 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.
• 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.
• 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.
• 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.

Study record dates

Study Major Dates

• Study Start (Estimated): April 1, 2026
• Primary Completion (Estimated): October 1, 2026
• Study Completion (Estimated): November 1, 2026

Study Registration Dates

• First Submitted: March 18, 2026
• First Submitted That Met QC Criteria: April 7, 2026
• First Posted (Actual): April 14, 2026

Study Record Updates

• Last Update Posted (Actual): April 21, 2026
• Last Update Submitted That Met QC Criteria: April 16, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: Heart Rate Variability; Cortisol; Breathwork; Coherent Breathing; Resonance Breathing; Maastricht Acute Stress Test (MAST)
• Additional Relevant MeSH Terms: Trauma and Stressor Related Disorders; Mental Disorders; Stress Disorders, Traumatic; Stress Disorders, Traumatic, Acute
• Other Study ID Numbers: APK.002.107.2024

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Anonymized individual participant data that underlie the results reported in the published article (including HRV parameters, cortisol levels, and psychological scale scores) will be made available from the corresponding author upon reasonable request. Raw physiological signal files will not be shared to protect participant privacy. Data will be provided in a standardized tabular format (e.g., Excel or CSV).
• IPD Sharing Time Frame: Beginning 6 months and ending 36 months following article publication.
• IPD Sharing Access Criteria: Data will be shared with qualified researchers who provide a methodologically sound proposal. Requests should be directed to the corresponding author (adam.siebieszuk@sd.umb.edu.pl). To gain access, data requestors will need to sign a data access agreement.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No