Breathwork and Stress: Investigating the Mechanisms of Action and Effectiveness of Breathing Interventions in Modulating the Psychophysiological Response to Acute Stress Test
Phase: NA
Conditions: Healthy Adult Participants, Acute Stress Reaction, Stress Biomarkers, Healthy
Sponsor: Medical University of Bialystok

1.​ Study Protocol 
 
Human Subjects Protection Review approval no.: APK.002.107.2024 
 
Note: This document is an English translation of the protocol approved by Bioethics 
Committee of the Medical University of Bialystok 
 
Date: 01.02.2026 
 
Official Title: Breathwork and stress: investigating the mechanisms of action and 
effectiveness of breathing interventions in modulating the psychophysiological response to 
acute stress. 
 
The following blinded clinical trial design focuses on evaluating the impact of a breathing 
method utilizing Breathwork (breathing exercises) and Mindfulness components on 
physiological and psychological stress levels in response to an acute stress test. The efficacy 
of coherent breathing will be compared with both a breathing sham (15 bpm) and a control 
condition in the form of spontaneous, undirected breathing. This design will allow for a more 
precise determination of whether the decisive therapeutic factor is a specific form of 
breathing or rather a general effect related to attention, rhythm, and participant expectations. 
The preliminary research hypothesis assumes that, compared to the sham intervention, 
performing the breathing method will significantly lower both physiological and 
psychological stress parameters. 
 
Background: 
Excessive and negative stress affects a significant portion of modern society. Long-term 
exposure to psychological stress leads to the development of allostatic load, which is 
associated with the disruption of the proper functioning of the autonomic nervous system 
(ANS) and the hypothalamic-pituitary-adrenal (HPA) axis, as well as significant emotional 
and cognitive dysregulation. Consequently, stress is a key risk factor for the development of 
both mental and physical illnesses, particularly cardiovascular diseases [1–5]. Chronic stress 
is associated with disturbances in sleep, cardiovascular function, metabolism, and immunity. 
In epidemiological studies, chronic stress has been linked to a significantly higher risk of 
depression, anxiety disorders, and cardiovascular diseases. Reduced heart rate variability 
(HRV), as a marker of allostatic load, serves as an independent prognostic factor for all-cause 
mortality [4–10]. 
One method that has gained popularity in recent years as a stress reduction tool is 
breathwork—the conscious regulation of the rhythm and depth of breathing [2,11,12]. 
Breathing techniques, often based on yoga or mindfulness traditions, are increasingly 
appearing in clinical interventions, mobile applications, and wellness practices. A growing 

body of research confirms their effectiveness in improving psychological well-being, 
reducing symptoms of anxiety and depression, and strengthening parasympathetic nervous 
system functioning. Despite the wide variety of techniques, the common denominator of their 
action appears to be a positive impact on the ANS and the synchronization of physiological 
rhythms, making breathwork a promising, non-pharmacological strategy for mental health 
prevention [2,13-17]. 
Coherent breathing (breathing at a frequency of approximately 5.5 breaths/min) is currently 
recognized as one of the most effective breathing methods for modulating autonomic nervous 
system (ANS) activity and improving heart rate variability (HRV). Through the 
synchronization of cardiorespiratory rhythms and baroreflex activation, coherent breathing 
leads to increased parasympathetic activity and promotes a state of physiological calm 
(RSA), as confirmed by numerous imaging and psychophysiological studies [2,18–23]. 
However, recent large-scale blinded randomized controlled trials indicate that differences in 
long-term effects between various breathing techniques may be smaller than previously 
assumed. Fincham et al. demonstrated a lack of significant differences between coherent 
breathing (5.5 bpm) and placebo breathing at a natural rate (12 bpm). Conversely, a 
systematic review by Bentley et al. suggests that it is not the tempo, but rather the structuring 
and conscious control of breath itself that may constitute the primary therapeutic factor. 
Additionally, research shows that different breathing rates (both slow and fast) lead to similar 
improvements in mental health over the long term [19,21,24]. 
In light of these findings, it becomes crucial to empirically investigate which of these two 
potential mechanisms—specific physiological properties or a general psychological 
effect—plays a decisive role in the effectiveness of breathing techniques used in stressful 
situations. 
Study Objectives: 
The objective of this study is an in-depth analysis of the mechanisms underlying the 
effectiveness of breathing techniques in reducing responses to acute stress. Specifically, the 
study aims to determine whether the observed effects of breathing interventions result from a 
specific and key physiological parameter in breathwork—the optimal breathing rate (5.5 
breaths/min in coherent breathing), which, according to current research, maximizes 
baroreflex activation and cardiorespiratory synchronization—or whether a psychological 
component related to the act of performing a structured, relaxing activity plays the key role. 
The preliminary research hypothesis assumes that, compared to the sham intervention, 
performing the breathing method will significantly lower both physiological and 
psychological stress parameters. 
A significant aspect of this research experiment is ensuring uniform perceptual conditions 
across all interventions. To avoid potential suggestions regarding the superiority of one 
stress-management method over another—which could lead to so-called performance 
bias—it is crucial to present each technique appropriately to the study participants. In the 

information materials, both strategies will be presented as equivalent and widely used stress 
regulation methods that can yield beneficial effects. Such an approach will satisfy the 
requirement for cognitive blinding, thereby increasing the internal validity of the study 
[25,26]. 
Inclusion Criteria: 
The study will ultimately include up to 120 healthy, professionally active individuals or 
university students aged 18–60 (60 females and 60 males). 
Exclusion Criteria: 
Severe chronic diseases, including metabolic (diabetes) and psychiatric disorders; history of 
myocardial infarction or stroke; history of cardiac surgery; regular use of medications such as 
anxiolytics or beta-blockers (excluding hormonal contraception); pregnancy; participation in 
other scientific experiments; previous extensive experience with breathing techniques or 
regular independent breathing/meditation practice; professional athletes; Raynaud’s disease. 
Method of Physiological and Psychological Stress Induction: 
A vital component of the experiment is a valid, effective, and, most importantly, fully safe 
method of inducing stress in study participants. Psychophysiological responses to distress can 
be reliably induced under laboratory conditions using stress induction protocols. To this end, 
participants will be subjected to the Maastricht Acute Stress Test (MAST) protocol, which 
is designed as a simple, rapid, and non-invasive procedure aimed at activating the human 
stress response system. MAST is a reliable method for inducing robust autonomic, 
glucocorticoid, and subjective stress responses. The MAST protocol combines physical stress 
induction, unpredictability, uncontrollability, and the social-evaluative nature characteristic of 
other stress induction protocols [27,28]. 
The main components of the MAST protocol include: 
●​ Introductory Presentation: A brief 5-minute presentation familiarizing participants 
with the conditions and procedure of the test. 
●​ Video Recording: A dummy camera will be used; in reality, participants will never 
be recorded. This element is intended to evoke an additional social-evaluative effect 
reflecting realistic conditions, such as public speaking. 
●​ Main Phase (10 minutes): Alternating exposure to cold (hand immersion in cold 
water) and the instruction to perform mental arithmetic aloud (subtracting two-digit 
numbers from four-digit numbers). Both stress stimuli will have interval durations not 
exceeding 90 seconds, of which participants will be notified during the introductory 
presentation. 
Study Groups and Interventions: 

Through block randomization stratified by sex, participants will be assigned to three equal 
groups: control, sham, and experimental. The only difference between the groups is the 
breathing rate. A detailed description of the experimental protocol is provided below. 
Stress Management Methods Used in the Experiment (Research Interventions): 
●​ Coherent Breathing (~6 breaths per minute, 5.5s inhale / 5.5s exhale): An active 
intervention with documented physiological benefits utilizing slow, conscious 
diaphragmatic breathing. Participants will practice diaphragmatic breathing for 10 
minutes at a resonance frequency of ~0.1 Hz, guided by auditory cues, while focusing 
on the sensations accompanying the ventilation process. 
●​ Sham Breathing (15 breaths per minute, 2s inhale / 2s exhale): A structural 
placebo, devoid of deep physiological impact but maintaining the form of a conscious 
intervention. The breathing rate in the sham intervention corresponds to the 
spontaneous breathing rate. This will also be performed for 10 minutes. 
●​ Spontaneous Breathing (No instructions): A control condition reflecting the 
everyday way of responding to stress. Participants in the control group will be asked 
to close their eyes and attempt to relax, without any instructions regarding breath 
modulation. 
This study design allows for determining whether the specificity of the intervention (e.g., rate 
and depth of breath) indeed determines the strength of the physiological stress response, or 
whether this effect can be attributed to more general psychological mechanisms such as a 
sense of control, expectations, and focus of attention. 
 
General Study Design: 
Each participant will undergo a remote one day preparation phase and an in-person 
experimental phase on the third day: 
1.​ Preparation Phase (Remote): Participants will receive the following: a basic 
demographic questionnaire, the DASS-21 (21-item Depression Anxiety and Stress 
Scale [29–32]), and information materials regarding the breathing method used in the 
experimental phase. Participants will be required to complete both questionnaires 
once, review the information materials, and perform a 10-minute breathing protocol 
not used in the actual study (breathing at a rate of 3s inhale / 3s exhale). 
2.​ Experimental Phase (In-person): Participants will be seen at the research center 
(Department of Physiology, Medical University of Bialystok), where they will 
undergo a research protocol sequence involving two research interventions. To avoid 
natural cortisol fluctuations, appointments will be scheduled between 11:00 AM and 
7:00 PM. Participants will be informed in advance of the required 24-hour abstinence 
from alcohol or other psychoactive substances, as well as a minimum 3-hour break 

from caffeine (also known by other names such as theine, guaranine, or mateine), 
physical exercise, nicotine intake, or consuming anything other than water. 
 
Physiological and Psychological Assessments: 
The following measurements will be conducted during the experimental phase: 
●​ Salivary cortisol concentrations: Analyzed using an ELISA (Enzyme-Linked 
Immunosorbent Assay) kit. Samples will be collected using Salivette® Cortisol tubes 
with synthetic swabs. 
●​ RR intervals (HRV): Measured using a POLAR H10 heart rate monitor paired with 
the Elite HRV mobile application [33,34] as well as KubiosHRV mobile application. 
●​ Blood pressure and heart rate: Measured using an automated sphygmomanometer. 
●​ Current subjective stress levels: Assessed using Visual Analogue Mood Scales 
(VAMS) [35]. 
●​ State and Trait Anxiety: Measured using the State-Trait Anxiety Inventory (STAI) 
[36–39]. 
 
Experimental Phase Protocol: 
Regardless of the group assignment, the research protocol follows an identical structure, with 
the only variation being the specific stress-management method applied during the 
intervention. The sequence of the research protocol is as follows: 
1.​ Baseline (BASE): Upon arrival, the participant will be led to a neutral room to 
complete the necessary documentation and prepare for the study (fitting the Polar H10 
belt, rinsing the mouth with water). Baseline measurements of physiological and 
psychological stress parameters will then be conducted: completion of both parts of 
the STAI questionnaire; saliva sample collection; measurement of blood pressure, 
heart rate; subjective stress assessment on VAMS scales; and a 5-minute HRV 
measurement. 
2.​ First Research Intervention: The participant will be asked to perform the 10-minute 
breathing/sham/control method according to the instructions provided during the 
preparation phase. The researcher will leave the room during this time. 
3.​ Transfer: The participant will be moved to a second room specifically prepared for 
the MAST protocol. After the participant is seated, the researcher will ask them to 
sign a non-binding consent form for video and audio recording (participants will not 
actually be recorded). 
4.​ Pre-Stress (PRE): The researcher will begin the MAST preparation phase, where the 
tasks will be explained via a graphical presentation. Following the presentation, 
"PRE" measurements will be taken: subjective stress on VAMS scales; saliva sample 

collection; measurement of blood pressure, heart rate; and completion of the "State" 
portion of the STAI questionnaire, and a 5-minute HRV measurement. 
5.​ Acute Stress Phase (MAST): The participant will undergo the main 10-minute 
portion of the MAST protocol. This phase involves alternating hand immersion in 
cold water (4.5-5.5°C) and counting backward aloud, starting from a random 
four-digit number. 10-minute HRV measurement will be done during the task. 
Stimulus 
Presentation 
HI 
MA 
HI 
MA 
HI 
MA 
HI 
MA 
HI 
Duration 
5 min 
90 s 
45 s 
60 s 
60 s 
60 s 
90 s 
45 s 
60 s 
60 s 
 
6.​ Post-Stress (POST): Before the official end of the MAST protocol (the participant is 
only informed of a "short break" in testing), the researcher will perform "POST" 
measurements: VAMS scales, saliva collection, blood pressure, heart rate, the STAI 
"State" subscale, and a 5-minute HRV measurement. 
7.​ Debriefing: Immediately after data collection, the researcher will inform the 
participant that there is no break, the MAST test has officially concluded, and that 
they were not actually recorded. The participant is then moved back to the first neutral 
room. 
8.​ Second Research Intervention: The participant will again perform the 10-minute 
breathing/sham/control method. The researcher will leave the room and start 
10-minute HRV recording. 
9.​ End of Intervention (END): A 5-minute final measurement will be conducted, 
collecting all parameters again: VAMS scales, saliva collection, blood pressure, heart 
rate, and a 5-minute HRV measurement. 
10.​Recovery: After the "END" measurement, the participant will be asked to wait an 
additional 10 minutes in a seated position. This period accounts for the delayed 
glucocorticoid response, as salivary cortisol levels typically peak approximately 15 
minutes after exposure to a stressor. 
11.​Follow-up (FOLLOW): The final stage involves a comprehensive reassessment of 
stress parameters: STAI "State" subscale; saliva collection; blood pressure, heart rate; 
VAMS scales; and a final 5-minute HRV measurement. 
 
Fazy 
badania 
Control Group 
Sham Group 
Experimental Group 
1 
Preparation + Baseline 
measurement (BASE) 
Preparation + Baseline 
measurement (BASE) 
Preparation + Baseline 
measurement (BASE) 

2 
Intervention I – Control 
(Spontaneous breathing) 
+​ DURING1 
measurement 
Intervention I – Sham 
breathing 
+​ DURING1 
measurement 
Intervention I – Coherent 
breathing 
+​ DURING1 
measurement 
3 
Transfer to the second 
room 
Transfer to the second 
room 
Transfer to the second 
room 
4 
MAST presentation + 
PRE measurement 
MAST presentation + 
PRE measurement 
MAST presentation + 
PRE measurement 
5 
MAST Test – Exposure 
to stress stimulus 
+​ STRESS 
measurement 
MAST Test – Exposure 
to stress stimulus 
+​ STRESS 
measurement 
MAST Test – Exposure 
to stress stimulus 
+​ STRESS 
measurement 
6 
POST measurement 
POST measurement 
POST measurement 
7 
Conclusion of test and 
transfer to the first room 
Conclusion of test and 
transfer to the first room 
Conclusion of test and 
transfer to the first room 
8 
Intervention II – Control 
(Spontaneous breathing) 
+​ DURING2 
measurement 
Intervention II – Placebo 
breathing 
+​ DURING2 
measurement 
Intervention II – 
Coherent breathing  
+​ DURING2 
measurement 
9 
Final measurement 
(END) 
Final measurement 
(END) 
Final measurement 
(END) 
10 
Recovery phase 
Recovery phase 
Recovery phase 
11 
FOLLOW 
measurement 
FOLLOW 
measurement 
FOLLOW 
measurement 
 

The following parameters will be evaluated as part of the analysis: 
1.​ STAI (State-Trait Anxiety Inventory) scores: Assessment of state and stress anxiety 
levels. 
2.​ Cortisol Analysis: Salivary cortisol levels as a primary biomarker of the endocrine 
stress response. 
3.​ Subjective Stress (VAMS): Intensity of perceived stress as measured on the Visual 
Analogue Mood Scale from 0 to 100 in terms of perceived: 
○​ Stress 
○​ Relaxation 
○​ Mental tension 
○​ Calmness 
HRV (Heart Rate Variability) Parameters including: 
●​ RMSSD (Root Mean Square of Successive Differences): A time-domain measure of 
HRV reflecting parasympathetic nervous system (PNS) activity. 
●​ SDNN (Standard Deviation of NN intervals): An overall measure of heart rate 
variability. 
●​ Kubios PNS Index: A composite indicator of parasympathetic nervous system 
activity. 
●​ Kubios SNS Index: A composite indicator of sympathetic nervous system activity. 
●​ LF Absolute Power: Low-frequency power, reflecting the balance between the 
sympathetic and parasympathetic systems. 
●​ HF Absolute Power: High-frequency power, primarily associated with 
parasympathetic (vagal) activity. 
●​ LF/HF Ratio: The ratio of low-frequency to high-frequency power, used as an index 
of autonomic balance. 
●​ Mean RR: The average duration of RR intervals. 
●​ Mean HR: Average heart rate. 
Blood Pressure Parameters: 
●​ SBP (Systolic Blood Pressure): The pressure in the arteries during heart contraction. 
●​ DBP (Diastolic Blood Pressure): The pressure in the arteries during the heart's 
relaxation phase. 

      While a wide range of psychophysiological and biochemical markers is recorded 
according to the protocol, the final statistical analysis and primary publication may focus on a 
subset of the most relevant parameters to ensure clarity and avoid redundancy. All collected 
heart rate variability (HRV) parameters may undergo standard mathematical transformations 
(e.g., natural log transformation) where necessary to meet the assumptions of parametric 
statistical testing and ensure the validity of the results. 
  ​
The primary objective of this analysis is to elucidate how chronic stress and heart rate 
variability influence psychophysiological outcomes. Furthermore, the study aims to 
determine the interdependencies between psychological test results, HRV parameters, cortisol 
levels, glucose levels, and subjective stress perception. 
 
·         Polish, validated version of the DASS-21 (21-item Depression Anxiety and Stress 
Scale) questionnaire.  [29–32]: 

 
 

 
Polish, validated version of the STAI (State-Trait Anxiety Inventory) questionnaire 
(STAI-S and STAI-Y subscales). [39]: 
 English versions: 
 

 
 
 
  
  
 

·          VAMS (Visual Analogue Mood Scales) for assessing subjective mood (stress, 
relaxation, mental tension, calmness/tranquility).[35]: 
 
 
 

 
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Podręcznik. Pracownia Testów Psychologicznych Polskiego Towarzystwa Psychologicznego 2002. 
 
 
2.​ Informed Consent Form 
 
INFORMED CONSENT FORM FOR STUDY PARTICIPATION (TRANSLATED) 
Title: "Breathwork and stress: investigating the mechanisms of action and effectiveness of 
breathing interventions in modulating the psychophysiological response to acute stress." 
Full Name: …………………………………………….................................. 
I hereby provide my consent to participate in a study aimed at evaluating the effectiveness of stress 
reduction methods utilizing breathing exercises. The objectives, conditions of participation, potential 
risks and benefits, as well as all procedures and stages of the research protocol, have been explained 
to me. I also confirm that I have been informed of the opportunity to ask questions regarding this 
medical experiment. I understand that I may withdraw my consent at any stage of the study without 
providing a reason and without facing any adverse consequences or claims. I declare my consent for 
the processing of my clinical data by the individual conducting the study, provided that my personal 
data is protected and all information obtained during the study is treated as confidential. 
Białystok, date: …………….……………………………… 
Participant’s Signature .................................................... 
Researcher’s Signature .................................................... 
 

 
3.​ Statistical Analysis Plan 
 
Data Preparation and Normality Testing: The normality of the data distribution for all 
physiological and psychological variables will be assessed using the Shapiro-Wilk test. For 
any parameters demonstrating a non-normal distribution, a natural log transformation (ln) 
will be applied prior to parametric testing to meet the necessary assumptions for Analysis of 
Variance (ANOVA). 
Data Integrity and Quality Control: To ensure high data quality and minimize loss due to 
technical issues, HRV data is recorded simultaneously using two mobile applications: Elite 
HRV and Kubios HRV, both connected to a Polar H10 monitor. This redundancy is 
implemented because pilot testing revealed occasional freezing of the Kubios HRV app; in 
such instances, identical data segments will be recovered from the Elite HRV backup. 
HRV Exclusion Criteria: For the 5-minute static HRV measurement windows, a minimum of 
4 minutes of valid RR interval data is required. This 1-minute buffer accounts for potential 
movement artifacts (e.g., chest strap displacement) or software synchronization errors. 
Participants with recordings shorter than 4 minutes in any primary measurement window will 
be excluded from the final HRV analysis to ensure the physiological validity of the results. 
Statistical analysis: 
1.​ Primary Statistical Model (Discrete Points): A two-way mixed-design Analysis 
of Variance (ANOVA) will be utilized to analyze changes across the five primary 
5-minute measurement points: BASE, PRE, POST, END, and FOLLOW. The model 
will include Intervention Group (Coherent, Sham, Control) as the between-subjects 
factor and Time as the within-subjects factor. 
 
2.​ Secondary Statistical Analysis (Process Windows): To evaluate the immediate 
psychophysiological dynamics, separate statistical analyses will be conducted for the 
three 10-minute continuous monitoring windows: First Intervention (DURING1), 
Acute Stress Phase (STRESS), and Second Intervention (DURING2). These 
analyses will specifically: 
●​ Verify the efficacy of the MAST stressor by comparing SNS Index and Heart Rate 
across groups during the STRESS window. 
●​ Assess the "buffering" effect of coherent breathing by analyzing RMSSD and PNS 
Index during the DURING1 and STRESS windows compared to sham and control 
groups. 
●​ Confirm protocol adherence using the Kubios RESP algorithm to compare 
respiratory rates between the 6 bpm and 15 bpm groups during both intervention 
windows. 
Post-hoc Analysis: To explore significant main effects or Group x Time interactions, 
Tukey’s HSD post-hoc test will be employed. This will allow for multiple pairwise 

comparisons between groups at specific time points and within groups across the study 
phases to identify where significant differences occur. The level of statistical significance for 
all tests is set at p < 0.05. 
Biochemical Analysis (Technical details): Salivary cortisol concentrations are determined 
using ELISA kits (Abcam). The absorbance of the samples is measured using a BayoTec 
microplate reader. All samples will be analyzed in accordance with the manufacturer's 
instructions.