Breathing Interventions for Relaxation: Dosing Through Extended Exhale
Phase: NA
Conditions: Healthy Adults, Stress
Sponsor: Vanderbilt University Medical Center

 
 
 
Revision Date: 10/15/18 
 
1 
 
 
 
 
 
 
 
 
 
 
 
 
 
BREATHING INTERVENTIONS FOR RELAXATION: DOSING 
THROUGH EXTENDED EXHALE AMONG HEALTHYADULTS 
 
Principal Investigator: 
Alfredo Gamboa, M.D. 
Associate Professor, Clinical Pharmacology  
Vanderbilt University Medical Center  
Nashville, TN 37232-8300 
 
Supported by: 
National Center for Complementary and Integrative Health 

 
 
 
Revision Date: 10/15/18 
 
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TABLE OF CONTENTS 
Page 
STUDY TEAM ROSTER............................................................................................................. 1 
PARTICIPATING STUDY SITES ................................................................................................ 1 
PRÉCIS ........................................................................................................................................... 2 
1. STUDY OBJECTIVES ............................................................................................................. 3 
2. BACKGROUND AND RATIONALE .................................................................................... 4 
2.1 
Background ..................................................................................................................... 4 
2.2 
Study Rationale ............................................................................................................... 4 
3. STUDY DESIGN ....................................................................................................................... 5 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS ................................................ 5 
4.1 
Study Sample Selection .................................................................................................. 5 
4.2 
Study Enrollment Procedures ......................................................................................... 6 
5. STUDY INTERVENTIONS .................................................................................................... 8 
5.1 
Interventions, Administration, and Duration .................................................................. 8 
5.2 
Concomitant Interventions .............................................................................................. 9 
5.2.1 Allowed Interventions ................................................................................................. 9 
5.2.2 Required Interventions ................................................................................................ 9 
5.2.3 Prohibited Interventions .............................................................................................. 9 
5.3 
Adherence Assessment ................................................................................................... 9 
6. STUDY PROCEDURES ........................................................................................................ 11 
6.1 
Schedule of Evaluations ................................................................................................ 11 
6.2 
Description of Evaluations ............................................................................................ 12 
6.2.1 Screening Evaluation ................................................................................................ 12 
6.2.2 Enrollment, Baseline, and/or Randomization ........................................................... 12 
6.2.3 Blinding..................................................................................................................... 13 
6.2.4 Followup Visits ......................................................................................................... 13 
6.2.5 Completion/Final Evaluation .................................................................................... 13 
7. SAFETY ASSESSMENTS ..................................................................................................... 14 
7.1 
Specification of Safety Parameters ............................................................................... 14 
7.2 
Adverse Events and Serious Adverse Events ............................................................... 14 
7.3 
Reporting Procedures .................................................................................................... 15 
7.4 
Followup for Adverse Events ....................................................................................... 16 

 
 
 
Revision Date: 10/15/18 
 
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7.5 
Safety Monitoring ......................................................................................................... 16 
8. INTERVENTION DISCONTINUATION ........................................................................... 16 
9. STATISTICAL CONSIDERATIONS .................................................................................. 17 
9.1 
General Design Issues ................................................................................................... 17 
9.2 
Sample Size and Randomization .................................................................................. 18 
9.3  
Definition of Populations .............................................................................................. 18 
9.4 
Interim Analyses and Stopping Rules ........................................................................... 18 
9.5 
Outcomes ...................................................................................................................... 19 
9.5.1 Primary Outcome ...................................................................................................... 19 
9.5.2 Secondary Outcomes ................................................................................................ 20 
9.6 
Data Analyses ............................................................................................................... 21 
10. DATA COLLECTION AND QUALITY ASSURANCE .................................................. 22 
10.1 
Data Collection Forms .................................................................................................. 22 
10.2 
Data Management ......................................................................................................... 22 
10.3 Quality Assurance ............................................................................................................. 22 
10.3.1 
Training ................................................................................................................. 22 
10.3.2 
Metrics .................................................................................................................. 22 
10.3.3 
Protocol Deviations ............................................................................................... 23 
10.3.4 
Monitoring ............................................................................................................ 23 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY ................................................... 23 
11.1 
Institutional Review Board (IRB) Review .................................................................... 23 
11.2 
Informed Consent Forms .............................................................................................. 23 
11.3 
Participant Confidentiality ............................................................................................ 23 
11.4 
Study Discontinuation ................................................................................................... 23 
12. REFERENCES ...................................................................................................................... 24 

 
 
 
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13. SUPPLEMENTS/APPENDICES ........................................................................................ 27 
I. 
Consent Form 
II. 
Questionnaires 
a. Demographics 
b. Medications 
c. Perceived Stress Scale 
d. Positive and Negative Affect Schedule-X (PANAS-X) 
e. PROMIS Anxiety CAT Data Item Bank 
f. PROMIS Depression CAT Data Item Bank 
g. PROMIS Sleep Disturbance CAT Data Item Bank 
h. Epworth Sleepiness Scale 
i. Concomitant interventions 
III. 
Breathing visit fidelity documentation 
IV. 
Recruitment 
a. Flyer for recruitment 
b. Email and electronic message for recruitment 
 

 
 
 
Revision Date: 10/15/18 
 
1 
 
STUDY TEAM ROSTER 
 
Alfredo Gamboa, MD, Principal Investigator 
Vanderbilt University Medical Center  
Campus zip 6602 
Nashville, TN 37235-0002 
Tele: 615-343-3649 
Fax: 615-343-8649 
alfredo.gamboa@vanderbilt.edu 
 
Hui Nian, PhD, Co-investigator 
Vanderbilt University Medical center 
2525 West End, Ste. 110000 
Nashville, TN 37203 
Tele: 615.875.3004 
Fax: 615.343.4924 
hui.nian@vanderbilt.edu 
 
Kenneth Wallston, PhD, Co-investigator  
Vanderbilt University School of Nursing  
421 Godchaux Hall 
Nashville, TN 37235-0002 
Tele: 615-343-3317 
Fax: 615-343-5898 
kenneth.wallston@vanderbilt.edu 
 
Gurjeet Birdee, MD MPH, Former Principal Investigator 
Vanderbilt University Medical Center 
Campus zip 8300 
Nashville, TN 37235-0002 
Tele: 615-936-0584 
Fax: 615-936-1269 
gurjeet.birdee@vanderbilt.edu 
 
 
PARTICIPATING STUDY SITES 
Vanderbilt University Medical Center 
Campus zip 8300 
Nashville, TN 37235-0002 
Tele: 615-936-0584 
Fax: 615-936-1269 
 
 
 
 
 

 
 
 
Revision Date: 10/15/18 
 
 
 
 
PRÉCIS 
Breathing Interventions for Relaxation: Dosing Through Extended Exhale 
among Healthy Adults 
Objectives 
The objectives of this study are: 
Specific Aims R61 (n=100) 
1. Compare 12 weeks of slow breathing exhale>inhale vs. exhale=inhale on changes 
in physiological stress (primary outcome: heart rate variability; secondary outcomes: 
baroreflex sensitivity, catecholamines). 
2. Compare 12 weeks of slow breathing exhale>inhale vs. exhale=inhale on changes 
in psychological stress (primary outcome: anxiety; secondary outcomes: affect, 
arousal). 
3. Compute the correlations between changes in physiological and psychological 
outcomes (e.g., changes in heart rate variability correlated with changes in perceived stress). 
 
Design and Outcomes 
We propose a randomized study among 100 healthy adults comparing the effects 
on stress of two standardized slow breathing protocols derived from therapeutic 
breathing techniques from yoga (pranayama are yoga techniques related to 
breathing). In a 12 week study we will measure if exhale>inhale vs. exhale=inhale 
produces measurable and meaningful differences in stress among healthy adults. 
Participants will meet with mind-body therapists weekly to learn slow breathing, and 
asked to perform exercises daily for 12 weeks. Data will be collected at baseline, 6 
and 12 weeks. There will be an optional questionnaire follow-up at 26 weeks. 
Interventions and Duration 
A. Intervention: Participants will be randomized to 12 weeks of slow breathing 
with either exhale>inhale versus exhale=inhale. Each week for 12 weeks, the 
participant will meet with the mind-body therapist for a 45 minute visit. 
Participants will be taught the breathing interventions in a private office on 
the Vanderbilt University campus. 
B. Duration: Participants will be enrolled into 12 weeks of the interventions 
and have outcome measures assessed at baseline, 6 weeks, and 12 weeks. 
Participants may elect to be contacted for a 26 week follow-up questionnaire. 
 
 
Sample Size and Population 
A total of 100 healthy adults, age 30-60 years will be enrolled in this study. Each 
participant will be randomized using a 1:1 ratio stratified by gender. 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Revision Date: 10/15/18 
 
 
 
 
1. STUDY OBJECTIVES 
 
Specific Aim 1: Compare 12 weeks of slow breathing exhale>inhale versus 
exhale=inhale on changes in physiological stress (primary outcome: heart rate 
variability, secondary outcomes: baroreflex sensitivity, catecholamines). 
 
Primary hypotheses: We hypothesize that participants with exhale>inhale versus 
exhale=inhale will have higher increases in parasympathetic tone as measured by the 
high frequency component of heart rate variability (HF HRV). 
Secondary hypotheses: 
1. We hypothesize that participants with exhale>inhale versus exhale=inhale will have higher 
increases in parasympathetic tone as measured by baroreflex sensitivity alpha index. 
2. We hypothesize that baseline parasympathetic tone will modify response to breathing 
interventions. Specifically, we hypothesize that participants with lower parasympathetic tone 
at baseline will have the greater increases in parasympathetic tone as measured by HF HRV 
and BRS alpha index. 
 
Specific Aim 2: Compare 12 weeks of slow breathing exhale>inhale versus 
exhale=inhale on changes in psychological stress (primary outcome- anxiety, secondary 
outcomes: affect, arousal). 
 
Primary hypotheses: We hypothesize that participants with exhale>inhale versus 
exhale=inhale will have further decreases in psychological stress as measured by the 
PROMIS Anxiety. 
 
Secondary hypotheses: 
1. We hypothesize that participants with exhale>inhale versus exhale=inhale will have further 
decreases in stress as measured by Anxiety Sensitivity Index-3 (ASI-3), Beck Anxiety 
Inventory, Body Vigilance Scale, Panic Disorder Severity Scale, and Perceived Stress Scale-
10 (PSS) and decreases in negative affect as measured by PROMIS Depression scale.and 
Positive and Negative Affect Schedule-X (PANAS-X). 
2. We hypothesize that baseline psychological stress will modify response to breathing 
interventions. Specifically, we hypothesize that participants with higher stress at baseline 
will have the further decreases in stress as measured by the PROMIS Anxiety scale. 
Exploratory research questions: We consider arousal as an exploratory measure in this 
proposal. In our pilot, exhale>inhale versus exhale=inhale had improvements in 
daytime arousal as measured by the Epworth Sleepiness Scale. For the present proposal, 
we will examine differences in arousal from baseline, 6, to 12 weeks as measured by the 
Epworth Sleepiness Scale. We will also explore the relationship between baseline sleep 
disturbance as measured by PROMIS Sleep Disturbance and changes in arousal. 
 
Specific Aim 3: Compute the correlation between physiological and psychological 
changes (e.g. changes in heart rate variability correlated with changes in PROMIS 
Anxiety). 
Primary hypotheses: We hypothesize that changes in physiological and psychological 
measures will be moderately correlated. 
 

 
 
 
Revision Date: 10/15/18 
 
 
2. BACKGROUND RATIONALE 
 
2.1 
Background on Condition, Disease, or Other Primary Study Focus 
Deep and slow breathing exercises are commonly used by individuals as a means to 
relax. According to the National Health Interview Survey, 12.5% of adults in the U.S. report 
using deep breathing exercises for health.1 As it relates to health, breathing exercises are 
categorized as a mind-body practice.2 There have been various clinical applications of slow 
breathing including treatment of stress-related disorders (anxiety, depression, acute or  
chronic pain), cardiovascular diseases (hypertension, heart failure), and pulmonary diseases 
(asthma, chronic obstructive lung disease).3 Slow breathing is an integral part of most mind- 
body practices including yoga, t’ai chi, qi gong, meditation techniques (Zen, Transcendental 
Meditation, Vipassana, Mindfulness-Based Stress Reduction), and other relaxation  
techniques (relaxation response, biofeedback). The clinical efficacy of slow breathing for 
these conditions is largely unproven. Since slow breathing is a common component to many 
mind-body practices, it is important to understand the potential role of slow breathing 
exercises on human health. 
Research has demonstrated that slow breathing produces changes in the autonomic 
nervous system.4 In most studies, slow breathing has been defined as a respiratory rate of 
<10 breaths/min. Slower respiratory rates have been shown to decrease sympathetic and 
increase parasympathetic tone. This may be partially mediated through alteration of intra-
thoracic pressures,5-7 stimulation of arterial and cardiopulmonary baroreceptors8,9 and 
afferent pulmonary stretch receptors.6,10 There has been a particular focus on a respiratory 
rate of 6 breaths/min (0.10 Hz) where cardiopulmonary entrainment occurs.11,12 Deep 
breathing exercises performed at 0.10 Hz produce increased baroreflex sensitivity and 
decreased sympathetic nerve activity as compared to spontaneous or regulated breathing at 
rest respiratory rates (~0.15 Hz).13,14 
To date, researchers have assumed that a low respiratory rate is chiefly responsible for 
inducing physiological and psychological relaxation. However, based on wide variety of 
mind-body practices, respiratory rate is not the single factor that produces relaxation during 
slow breathing. Practitioners modify the ratio of inhalation and exhalation to enhance 
relaxation. Specifically, extension of exhalation relative to inhale is believed to augment 
physiological and psychological relaxation. 
 
 
2.2 
Study Rationale 
There have been few studies examining if exhale>inhale versus exhale=inhale while 
breathing slowly augments physiological and psychological relaxation. All of these studies 
have examined acute effects from slow breathing. Most have focused on slow breathing at a 
respiratory rate of 6 breaths a minute.15 16 17 Results from these studies are mixed. Some have 
reported exhale>inhale versus inhale=exhale produces more physiological relaxation,15,17 
whereas others report no significant differences between groups.16 Only one study examined 
the acute effect of different breath ratios on physiological and psychological stress with 
exhale>inhale decreasing both more than exhale=inhale.17 We collected preliminary data in 
a small randomized 6 week study among 21 healthy adults 
comparing the effects of two standardized slow breathing protocols on stress. 
We randomized participants to a daily practice of: (1) exhale greater than inhale in length 

 
 
 
Revision Date: 10/15/18 
 
 
(exhale>inhale, goal ratio 2:1) versus (2) exhale equal to inhale in length (exhale=inhale). 
That pilot demonstrated our ability to recruit and retain participants and to have them 
complete physiological and psychological stress measures at designated time points. 
Although the exhale>inhale group showed trends towards more relaxation compared to the 
exhale=inhale group, the study had insufficient power to measure definitive differences 
between the two groups. The rationale of the present study is to build on our preliminary 
data with by conducting a larger (n=100) and longer (12 week) randomized study to assess 
if exhale>inhale vs. exhale=inhale produces measurable and meaningful differences in stress 
among healthy adults. 
 
3. STUDY DESIGN 
We propose a prospective, randomized, double-blinded, study among healthy patients to 
compare the effects of exhale>inhale versus exhale=inhale on physiological and 
psychological stress. In a sample of 100 participants we will compare the clinical 
effectiveness of a 12-week program of slow breathing exercises, consisting of exhale>inhale, 
to a 12-week program of slow breathing exercises, consisting of exhale=inhale. Outcome 
assessment will occur at baseline, 6 and 12- weeks for: physiological stress (autonomic tone: 
heart rate variability, baroreflex sensitivity), and psychological anxiety (PROMIS Anxiety 
scale, Anxiety Sensitivity Index-3, Beck Anxiety Inventory, Body Vigilance Scale, Panic Disorder 
Severity Scale, and Perceived Stress Scale-10), affect (PROMIS depression Scale, PANAS-X), and 
arousal (Epworth Sleepiness Scale, PROMIS Sleep Disturbance Scale]. Psychological 
measures will be repeated at 26 weeks after study completion as follow-up. The primary 
outcomes of the clinical trial will be heart rate variability measured by spectral analysis 
and psychological stress measured by the PROMIS Anxiety. 
Randomization will occur after baseline testing and assessment of initial compliance to 
the intervention. Treatment assignments will be generated in a 1:1 ratio stratified by 
gender.Participants will be randomized to 12 weeks of slow breathing with either 
exhale>inhale versus exhale=inhale. Each week for 12 weeks, the participant will meet with 
the mind-body therapist for a 45 minute visit. Participants will be taught the breathing 
interventions in a private office on the Vanderbilt University campus. 
 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS 
 
4.1 
Study Sample Selection 
All participants enrolled will meet the inclusion and exclusion criteria of the study as stated 
in Table 1. PROMIS Anxiety scale will be administered during the screening phase. Only 
participants with a PROMIS Anxiety score of 45 or higher will be eligible (half a stand 
deviation below the mean or higher for the general population). We will also evaluate 
compliance during the screening phase. We will only include those who demonstrate high 
compliance to the intervention during the screening phase by practicing at least 3 times a 
three weeks. Secondly, participants must be able to achieve a respiratory rate of ≤8 
breaths/minute but not ≤3 breaths/minute.  
Efforts will be made to reduce the length of time between the screening phase and 
enrollment. However, if baseline measures show that, in the time between these events, the 

 
 
 
Revision Date: 10/15/18 
 
 
participant no longer meets the inclusion criteria (as listed in Table 1) then participant 
eligibility may be reassessed. If the participant is removed from study for this reason it will 
be before the first intervention occurs. 
 
Subjects must have the ability to understand study procedures and comply with them for the 
entire length of the study. For men and women of reproductive capability, contraception will 
not be necessary or required. 
 
We will not exclude patients on the basis of gender, race, ethnicity, or religious preferences 
and practices. 
 
Table 1. Sample Selection Criteria 
Inclusion 
• 
Age 30 to 60 years 
• 
After two weeks of intervention: 
• 
Breathe 8 or less a minute while practicing 
• 
Not breathe 3 or less breaths a minute while practicing 
• 
Practiced 3 or more times a week 
• 
English speaking 
• 
PROMIS Anxiety scale ≥ 45 
Exclusion 
• 
Hypertension 
• 
Heart disease: history of coronary artery disease, myocardial 
infarction, significant valvular disease, or congestive heart failure 
• 
Diabetes 
• 
Renal disease 
• 
Anxiety disorder 
• 
Depression 
• 
Other psychiatric conditions including schizophrenia or bipolar 
disorder 
• 
Attention-deficit-disorder or Attention-deficit-hyperactivity 
disorder 
• 
Musculoskeletal condition limiting capacity to perform simple 
movements such as chronic lower back pain or neck pain 
• 
Pulmonary disorder (asthma, chronic obstructive lung disease, 
obstructive sleep apnea) 
• 
Smoker 
• 
Currently 
taking 
blood 
pressure 
medications, oral diabetic medication or 
insulin 
• 
Current participation in a mind-body 
practice/program 
• 
Current cancer other than non- 
melanoma skin cancer 
• 
Regular swimmer 
• 
Plays wind or brass musical 
instruments 
• 
Pregnant 
• 
 
 
 
4.2 Study Enrollment Procedures 
Recruitment and Referral Sources: Participants will be recruited through flyers 
and research email lists. 
(1) Flyers: Flyers will be posted in common areas of Vanderbilt University and public 
places in Nashville (groceries stores, retail stores, or restaurants on public notice 
boards). See flyers in Appendix. 
(2) Research email lists: 
a. MyResearch: We will use MyResearch at Vanderbilt (MRAV) to email potential 
volunteers. MRAV is a repository of over 17,000 Vanderbilt patients that have 
opted in to be contacted directly by e-mail to participate in research or to provide 
input on research ideas. We will send an IRB approved study description through 
MRAV at 3 month intervals for recruitment during the enrollment period. See 
Appendix for sample email. 

 
 
 
Revision Date: 10/15/18 
 
 
VICTR Research Notification Distribution List: VICTR Research Notification Distribution 
List is a recruitment tool, available to Vanderbilt researchers that reaches over 18,500 
Vanderbilt faculty and staff, as well as members of the Middle Tennessee community. The 
email tool provides investigators a forum for advertising for volunteers for a specific study 
and the ever-growing distribution list allows all individuals interested in learning about 
research opportunities to subscribe to receive the email notifications. Email notifications are 
limited to 
IRB approved language, describe study specifics and provide contact information. Supported by 
VICTR, an ongoing marketing plan exists for retaining and increasing distribution list 
subscribers. To submit an ad, investigators complete an Email Notification Request Form. Ads 
are emailed to the distribution list within the next 5 business days. We will send an IRB approved 
study description through the list at 3 month intervals during the enrollment period. See 
Appendix for sample email. 
 
(3) Online websites: We will use ResearchMatch, an online recruitment tool hosted by Vanderbilt. 
ResearchMatch is a disease neutral online research recruitment and engagement platform that 
aims to remove the barriers to patient participation in research by partnering with researchers 
and advocacy groups to increase patient engagement and access to information across the 
research enterprise. The site is specifically designed to match individuals interested in research 
participation with researchers in their area and is funded in part by the National Center for the 
Advancement of Translational Science (NCATS), an office of the National Institute of Health 
(NIH). The system allows people from anywhere in the country to self-register and express an 
interest in participating in research studies. ResearchMatch then provides information about 
those volunteers to researchers. Researchers may register to use ResearchMatch with either 
feasibility or recruitment access. Feasibility access is limited in that it only allows researchers 
to view aggregate de-identified data. Researchers with recruitment access, however, are able to 
conduct a more targeted search using their study’s inclusion/exclusion criteria. 
We have applied for recruitment access to identify a list of potential volunteers. 
We will send an IRB-approved recruitment message through the secure 
ResearchMatch clearinghouse to each volunteer on the list. See Appendix for sample 
recruitment message. If a Volunteer responds, “Yes, I am interested!” ResearchMatch 
will release the volunteer’s contact information to the researcher who is then able to 
directly contact the volunteer and follows normal study protocol. The registry includes 
all ages, healthy and with health conditions. 
ResearchMatch was developed in 2009 by institutions affiliated with the Clinical 
and Translational Science Awards Consortium (www.ctsacentral.org) and is 
maintained at Vanderbilt University. The Vanderbilt Institutional Review Board (IRB) 
provides oversight of the project as a recruitment tool. 
 
Enrollment Procedures: 
Individuals interested in participation will go through screening through REDCap to determine 
initial eligibility based on criteria listed above. If potentially eligible, research staff will 
contact the individual to explain the research protocol, answer questions, and discuss informed 
consents.  Potential subjects interested in participation will be scheduled for a research visit 
with research staff for primary screening. Primary screening will consist of a direct interview 
by research staff to confirm eligibility. If potentially eligible, participants will complete an 
informed consent  to proceed with secondary screening (see Appendix I. Informed Consent for 
Screening).  

 
 
 
Revision Date: 10/15/18 
 
 
 
Secondary screening will consist of baseline outcomes assessments including administration 
of PROMIS Anxiety Scale, and 2-weeks of intervention to assess compliance.  
 
After secondary screening, eligible and willing individuals will complete a second informed 
consent to participate in the randomized clinical trial (see Appendix I. Informed Consent for 
clinical trial).  
 
For enrolled participants, randomization will occur after 2 weeks of basic breathing and 
assessment of breathing by mind- body teacher. Treatment assignments will be generated in a 
1:1 ratio stratified by gender.
5. STUDY INTERVENTIONS 
 
5.1 
Interventions, Administration, and Duration 
Development: We designed breathing protocols to examine the effect of slow breathing on 
stress measures among healthy adults. The breathing interventions for this study were 
developed by Dr. Birdee in conjunction with an expert panel of 3 mind-body therapists. The 
breathing protocol uses a well-developed progression of breathing taught in the 
Krishnamacharya tradition of yoga (Viniyoga).18,19, 20 
Breathing instruction: Participants will receive 12 weekly private classes taught by a 
certified mind-body teacher (yoga teachers) specifically trained to deliver the slow breathing 
protocols. Teachers are specifically instructed not to reveal the goal of the breathing exercises 
(i.e. relaxation). Each class will be 45 minutes long. The first two weeks of the protocol 
teaches same basic slow and deep breathing to both treatment arms. The purpose of the first 
two weeks is to allow all participants an opportunity to become familiar with basic 
components of deep breathing. Participants will be asked to perform breathing exercises daily 
in the evening. 
Participants will be given written instructions and drawings to guide home practice. 
During the two weeks for secondary screening, the teacher will assess the subjects’ 
respiratory rate while breathing deeply. Subjects will be eligible only if they achieve a 
respiratory rate of 8 breaths a minute or less. This is based on principles of pranayama that 
breath ratios above this respiratory rate are not useful. On the other end of the spectrum, 
subjects who are already able to breathe very slowly, defined as 3 breaths per minute or 
less, will not be included. Subjects will only be included who perform the practice 3 or 
more times a week, based on self-report, during the first two weeks. 
To facilitate breathing regulation and standardization between participants, participants 
will be given an MP3 player with a recorded track that made an audible sound every second 
(60 Hz). Subjects will be asked to count the length of each breath based on the recording. 
Subjects demonstrating eligible respiratory rates will proceed to the randomized breathing 
interventions described below. 
Breathing rates are assigned based on the assessment of deep breathing performed at 2 
weeks. This provides an opportunity to identify a comfortable, starting, slow breathing rate 
for each participant. After randomization, participants will follow a week to week progression 
based on a pre-specified protocol for 10 weeks. This allowed breathing rates to be matched 
week by week between treatment groups. 
 

 
 
 
Revision Date: 10/15/18 
 
 
5.1.1 Slow breathing with exhale=inhale: 
Participants randomized to slow breathing with equal inspiration and expiration 
received progressive increases in both inspiration and expiration over 4 weeks 
until they reach a goal breath length or longest comfortable breath. Goal breaths 
were assigned based on initial breath assessment. Prior to performing the 
breathing practice, participants performed a few standardized yoga movements. 
The purpose of movements was to prepare the subject to sit and focus on 
breathing. Subjects randomized to exhale=inhale will receive progressive 
increases in both inspiration and expiration until they reach a goal breath length. 
Goal breaths will be assigned based on initial breath assessment. Prior to 
performing the breathing practice, subjects will perform a set of standardized 
breathing practices. The purpose of these few breathing practices is to prepare the 
subject to sit and focus on breathing. 
 
5.1.2 Slow breathing with exhale>inhale: 
Participants randomized to slow breathing with prolonged exhale to inhale ratio 
received progressive increases in expiration relative to inspiration based on 
initial assessment until they reached a goal breath length or longest comfortable 
breath. Prior to performing the breathing practice, subjects in this exhale>inhale 
condition performed the same yoga movements as the exhale-inhale group. 
 
See Appendix for detailed breathing intervention protocol. 
 
5.2 
Concomitant Interventions 
 
5.2.1 
Allowed Interventions 
All subjects will continue with routine treatment of medical conditions including all 
medications. Changes in medications will be at the discretion of usual healthcare 
providers. Any changes in medications or other concomitant interventions initiated 
during the study period will be documented (See Appendix for Concomitant 
Intervention data collection). 
 
5.2.2 
Required Interventions 
No additional interventions are required except for those proposed for in 
study protocol. 
5.2.3 
Prohibited Interventions 
Subjects receiving will be asked to not participate in other mind-body programs 
for the duration of the study. 
 
5.3 
Adherence Assessment 
Fidelity measures: We will assess fidelity to breathing interventions through direct 
observation by the mind-body teacher, self-report, and biometric garments-
HEXOSKIN. 
(1) Observation Each week, mind-body teachers observe participants performing the 
practice along with an audible metronome to measure breath rate and length of exhale and 

 
 
 
Revision Date: 10/15/18 
 
 
inhale. Mind-body teachers evaluate participate engagement, quality of technique, and 
breathe rate and exhale/inhale length (See Appendix for mind-body fidelity tracking form).  
(2) Self-Report Each week, participants are asked how frequently they practiced at home 
by the mind-body teacher. 
(3) Remote biometric garment: Lastly, we will use biometric garments, HEXOSKIN, to 
ascertain remote adherence to home breathing practice. As described previously, 
HEXOSKIN accurately records respiratory parameters including breathing rate, length of 
exhale/inhale through thoracic and abdominal strain gauges embedded in the shirt. Data for 
is stored in a small unit worn in the shirt. Each participant will be asked to wear a 
HEXOSKIN shirt during practice for one week for two time periods between weeks 3 to 8 
and weeks 9 to 12. For 2-3 nights during each of these weeks the participant will be asked 
to keep the shirt on overnight to monitor sleep. Participants will return the shirt after one 
week, and data from the device will be downloaded for analyses. We will use VivoSense 
software with the Complex Respiratory Module to analyze breathing intervention fidelity 
(Vivonoetics, San Diego, CA).

 
 
 
Revision Date: 10/15/18 
 
 
 
6. STUDY PROCEDURES 
 
6.1 
Schedule of Evaluations (Tx, Treatment, Wk, Week) 
 
Activity 
Primary 
screening  
Secondary screening 
 
Clinical Trial 
 
(Day-21 to 
Day -1) 
(Day 0) 
Wk1 Wk2 Wk3 Wk4 
Wk 
5 
Wk6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11 
Wk 
12 
Wk 13 
 
 
Assessment/Intervention 
 
 
 
 
Research 
visit 1 
Tx 
Visit 
1 
Tx 
Visit 
2 
Tx 
Visit 
3 
 
Tx 
Visit 
4 
Tx  
Visit  
5 
Tx 
Visit 
6 
 
Research 
visit 2 
Tx 
Visit 
7 
Tx 
Visit 
8 
Tx 
Visit 
9 
Tx 
Visit 
10 
Tx 
Visit 
11 
Tx 
Visit 
12 
 
Research 
visit 3 
Inclusion/Exclusion 
Criteria 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Enrollment/informed 
Consent  
• 
Consent for 
screening 
• 
Consent for clinical 
 
 
   X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data collection 
Physiological stress 
Psychological stress 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
Commitment 
interventions/medications 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
Randomization 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
Breathing intervention 
visits 
 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
 
Breathing intervention 
assessment 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Adherence: self-reported 
frequency of breathing 
practice at home 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Adherence: Remote monitoring 
(1 week period) 
 
 
 
 
 
X 
 
X 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

 
 
 
Revision Date: 10/15/18 
 
 
6.2 
Description of Evaluations 
6.2.1 
Screening Evaluation  
 
Consenting Procedure 
Subjects eligible and willing to participate in will be given a written informed 
consents by the research staff.  There are two consents for this research project. The 
first consent is to participate in the secondary screening. The informed consent for 
screening asks participants to participate in a 2 week study consisting of baseline 
assessment and 2 intervention visits. The purpose of the secondary screening is to 
determine if the individual is eligible for the clinical trial.  
The second consent is to participate in a clinical trial consisting of attending 
intervention sessions once a week for 10 weeks and 2 research visits.  
Details of all study procedures will be provided, including purpose of the studies, 
content of classes and details of tests to be done, frequency of classes and time 
commitment, risks and benefits, and voluntary nature of the program. Any changes to 
research procedures or intervention will prompt revision of the consent forms, with 
subsequent review and approval from Vanderbilt IRB. A copy of the signed consents 
will be stored in a locked file cabinet located in the secured office of the principal 
investigator. 
 
Screening 
Individuals interested in participation will go through screening through REDCap 
to determine initial eligibility based on criteria listed above. If potentially eligible, 
research staff will contact the individual to explain the research protocol, answer 
questions, and discuss informed consents. Potential subjects interested in 
participation will be scheduled for a first research visit with research staff. Initial 
eligibility will be confirmed with direct interview. If eligible, partipants will be 
consented for secondary screening. Secondary screening will consist of baseline 
assessments and 2 weeks of intervention to assess compliance.  
6.2.2 
Enrollment, Baseline, and/or Randomization Enrollment 
Enrollment into the clinical trial will be defined by patient having met all 
screening criteria and signed the informed consent for participation in the clinical trial 
 
Baseline Assessments 
For participants who have completed primary screening for eligibility and 
aenrolled for secondary screening, baseline assessments will be performed to 
measure study outcomes. 
• Autonomic tone: We will estimate autonomic tone of participants by standard 
autonomic testing of cardiovagal and sympathoneural functioning including 
heart rate variability and baroreflex sensitivity. Subjects will have fasted the 
night before testing, and testing will occur at the same time of the day for 
each subject to account for temporal variations of autonomic tone. 
• Questionnaires: PROMIS Anxiety Scale, Anxiety Sensitivity Index-3 (ASI-3), 
Beck Anxiety Inventory, Body Vigilance Scale, Panic Disorder Severity Scale, 
and Perceived Stress Scale-10 (PSS), PROMIS Depression scale, and PANAS-
X. 
• Catecholamine Profile: Plasma norepinephrine and epinephrine levels will be 

 
 
 
Revision Date: 10/15/18 
 
 
measured at baseline in supine and standing positions. 
• Current Medications 
• Concomitant Interventions 
 
Randomization 
Randomization will occur after secondary screening is complete. Treatment 
assignments will be generated in a 1:1 ratio stratified by gender. 
 
6.2.3 
Blinding 
 
Blinding is summarized in the table below, and described in detail as 
follows: 
1. Participants: Participants will not be able to be blinded to the treatment 
interventions as this is a behavioral intervention. However, during 
recruitment, screening, and consent, research staff and mind-body 
therapists will not state the specific hypothesized function/effect of the 
treatment interventions- physiological or psychological relaxation. We will 
describe the intervention as “focused breathing” that is being researched in 
regards to effects on healthy, and that focused breathing has been shown to 
improve general well-being. Mind-body therapists have been trained to 
deliver the protocol without specifying the function of the treatment 
interventions.  
2. Mind-body therapists: Therapists delivering the treatment intervention 
will not be blinded to the treatment interventions. Therapists will be 
blinded to primary and secondary outcome measures of stress. Therapists 
will have access to biometric data regarding fidelity of breathing 
interventions at home to ensure compliance to the intervention during the 
study period. 
3. Outcome assessors: Research staff collecting physiological and 
psychological stress data will be blinded to treatment intervention 
assignments. A research staff member separate from those collecting stress 
measures will collect data regarding fidelity to breathing interventions. 
4. Data analysts/statistician: Data analysts including the statistician will be 
blinded to treatment assignments until data analyses are complete.  
5. Principal investigator: Principal investigator will be blinded to treatment 
assignments until data analyses are complete. 
 
BLINDING TABLE: 
 
Stake holder 
Intervention 
group 
assignment 
Primary 
Mechanistic 
Outcome Measure 
(Physiological 
stress) 
Secondary 
Mechanistic 
outcome measure 
(Psychological 
stress) 
Participants 
No 
Yes 
Yes 
Instructors 
No 
Yes 
Yes 
Outcome 
Assessors 
Yes 
No 
No 
Statistician 
Yes 
No 
No 

 
 
 
Revision Date: 10/15/18 
 
 
Principal 
Investigators 
Yes 
No 
No 
 
 
6.2.4 
Follow-up Visits 
o Once a week in a private office on the Vanderbilt University campus 
o Slow breathing with either exhale > inhale or exhale = inhale 
o Adherence to intervention: Measure subject adherence to study protocol 
including quality, duration, and breathing goals 
o Adverse events: Document frequency and severity of adverse events during 
the study 
o Fidelity of breathing by self-report and direct observation by mind-body 
teachers 
o At 6 weeks 
o Autonomic tone 
o Catecholamine Profile 
o Questionnaires: PROMIS Anxiety Scale, Anxiety Sensitivity Index-
3 (ASI-3), Beck Anxiety Inventory, Body Vigilance Scale, Panic 
Disorder Severity Scale, and Perceived Stress Scale-10, PROMIS 
Depression scale, and PANAS-X. 
o Current Medications 
o Concomitant Interventions 
o Two one week periods from weeks 3-6 and 7-12: 
o Fidelity measure using biometric garment- HEXOSKIN 
6.2.5 
Completion/Final Evaluation 
o At 12 weeks 
o Autonomic tone 
o Catecholamine Profile 
o Questionnaires: PROMIS Anxiety Scale, Anxiety Sensitivity Index-3 
(ASI-3), Beck Anxiety Inventory, Body Vigilance Scale, Panic 
Disorder Severity Scale, and Perceived Stress Scale-10, PROMIS 
Depression scale and PANAS-X. 
o Current Medications 
o Concomitant Interventions 
 
Participants who discontinue the study intervention early will require no specific 
evaluations. 
 
The reason for discontinuation will be documented. If research participants no longer 
want to participate in the research study, we will terminate themfrom the study. 
Participants terminated from the study will undergo no further monitoring or follow-
up once they have stopped the study intervention. 
 
7. SAFETY ASSESSMENTS 
This research protocol employs breathing techniques among healthy adults that 
involves low risk to human subjects. Subjects will be frequently asked about 
symptoms relating to adverse events or discomfort. While expected to be very 

 
 
 
Revision Date: 10/15/18 
 
 
rare, possible serious adverse events related to breathing practices may include: 
o cerebrovascular accident 
o life threatening arrhythmias (e.g. ventricular tachycardia) 
o myocardial infarction 
o sudden death 
Non-serious adverse events from breathing techniques may include 
o acute symptomatic episodes from problems with blood pressure, 
o anxiety 
o lightheadedness/dizziness 
o muscle cramps, 
o musculoskeletal injury, 
o respiration (shortness of breath or 
respiratory discomfort),  
Other theoretical risks from study tests include: 
o local risks associated with venipuncture blood draws 
There may be risks and side effects that are currently unknown and/or unanticipated. 
Because these risks are unlikely given previous behavioral studies mind-body therapy 
practices, and most are inherent risks within the general population and not solely 
due to study procedures, the potential benefits of this study to the individual and 
society reasonably outweigh the risks. 
 
7.1 
Specification of Safety Parameters 
We will monitor for all adverse events during the study. The Data Safety and 
Monitoring Board (DSMB) will monitor and review adverse events, and toxicities 
and events that may be related to the intervention. This will include verification that, 
when indicated, these events have been reported to the appropriate agencies (e.g. 
IRB) and that such reports have been made in a timely manner. The DSMB (TBN) 
will consist of a biostatistician, medical physician, clinical trials researcher, and clinical 
psychologist. Dr. Birdee will also review information on the completeness of data and 
the rate of patient accrual relative to the protocol timeliness. 
 
7.2 
Adverse Events and Serious Adverse Events 
Definition - An adverse event (AE) is any untoward medical occurrence in a subject 
temporally associated with participation in the clinical study or with use of the 
experimental agent being studied. An adverse finding can include a sign, symptom, 
abnormal assessment (laboratory test value, vital signs, electrocardiogram finding, 
etc.) or any combination of these. 
 
 
A Serious Adverse Event (SAE) is any adverse event that results in one or more of 
the following outcomes: 
• 
Death 
• 
A life-threatening event 

 
 
 
Revision Date: 10/15/18 
 
 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
A persistent or significant disability/incapacity 
• 
A congenital anomaly or birth defect 
• 
Important medical event based upon appropriate medical judgment 
 
 
Research staff and mind-body therapists will document and report solicited AEs 
including physical or psychological discomfort related to anxiety, blood pressure 
changes, depression, lightheadedness/dizziness, muscle cramps, musculoskeletal 
injury, or respiration. Unsolicited events will be captured through observation by 
research staff. Research staff will document unsolicited events and report AEs 
according to data safety monitoring plan. Events will be documented in the RedCap 
data entry system including date and time of event which will avoid double capture. 
Adverse Reporting: The Independent Monitoring Committee (IMC) will monitor and 
review adverse events, and toxicities and events that may be related to the 
intervention. 
 
a) Non-serious adverse events will be reviewed on a monthly basis by the IMC. The 
IMC will be notified of serious adverse events within 24 hours of occurrence and 
reviewed within 48 hours. 
b) Unexpected fatal or life-threatening AEs related to the intervention will be reported 
to the NCCIH Program Officer within 7 days. Other serious and unexpected AEs 
related to the intervention will be reported to the NCCIH Program Official within 15 
days. 
 
c) Anticipated or unrelated SAEs will be handled in a less urgent manner but will be 
reported to the Independent Monitor(s), IRB, NCCIH, and other oversight 
organizations in accordance with their requirements. In the annual AE summary, the 
Independent Monitor(s) Report will state that they have reviewed all AE reports. 
 
7.3 
Reporting Procedures 
Adverse events will be documented by research staff in RedCap as shown in 
Appendix for adverse event documentation. Research staff will notify Dr. Birdee 
of non-serious adverse events within 24 hours. Dr. Birdee will present all non-
serious adverse events to the IMC for monthly review. Research staff will notify 
Dr. Birdee of serious adverse events immediately. Dr. Birdee will notify the IMC 
within 24 hours of occurrence, who will review the event within 48 hours. If upon 
review the SAE is determined to be related to the intervention, Dr. Birdee will 
report the occurrence to the NCCIH program officer in 7 days for unexpected 
fatal or life threatening AEs and 15 days for other serious and unexpected AEs. 
After being informed, Dr. Birdee will verify the severity of all AEs. Dr. Birdee 
will initially assess if the AE is related to the study interventions as definitely, 
probably, possibly or unrelated. The IMC will review and confirm the relatedness 
and severity of AEs. AEs reports will be distributed to the IMC electronically via 
email. 
 
7.4 
Follow-up for Adverse Events 

 
 
 
Revision Date: 10/15/18 
 
 
AEs will be monitored by the IMC and Dr. Birdee for recurrences, resolution, 
and ongoing toxicities that may be related to the intervention. Documentation and 
reporting for follow-up AEs will occur according to reporting procedures 
described in Section 7.3. The duration for follow-up will be for the length of the 
study period while patients are enrolled in the study. If non-serious adverse events 
are related to the breathing practice, then the protocol will be modified to avoid 
adverse events. If the frequency of non-serious adverse events is higher than 
anticipated or alters the benefit risk ratio, the study investigators after conferring 
with IMC, will also modify the breathing protocol. Two or more specific adverse 
events among subjects will prompt modification of the breathing protocol. 
 
7.5 
Safety Monitoring 
This Human Subjects research study will undergo independent monitoring 
from an IMC, which is described in detail in the Data Safety Monitoring Plan 
(DSMP) Monitoring Committee under the guidelines provided by NCCIH. The 
IMC will meet prior to enrollment of subjects into studies. 
 
8. INTERVENTION DISCONTINUATION 
The intervention will be discontinued for a participant if: 1) the intervention 
is associated  with an adverse effect for a specific subject i.e. subject does not 
tolerate the intervention, 2) the subject no longer is interested or willing to 
receive the intervention or participate in the study,  or 
3) the subject’s healthcare provider no longer recommends that the patient 
receive the  intervention for medical reasons. The IMC and the principal 
investigator will review cases upon occurrence for discontinuation of 
intervention. 
This study will be stopped prior to its completion if: (1) the interventions are 
associated with adverse effects that call into question the safety of the 
intervention, or (2) difficulty in study recruitment or retention will significantly 
impact the ability to evaluate the study endpoints, or 
(3) any new information becomes available during the trial that necessitates 
stopping the trial;  or 
(4) other situations occur that might warrant stopping the trial. 
Participants will be followed with their permission if the study is 
discontinued. The duration of follow-up will be length of the proposed study. 
We will continue to document adverse events, medications, and vital signs 
during the follow-up period. 
Temporary discontinuation of the treatment may occur if the participants are 
absent due to travel/vacation, illness including hospitalization, non-compliance, 
or difficulties with transportation to sessions. Treatment will continue upon 
return to regularly scheduled sessions. We will continue to document adverse 
events and medications during temporary discontinuation.

 
 
 
Revision Date: 10/15/18 
 
 
 
Subjects may withdraw from the study at any time. Investigators may end 
the participation of the subject if they no longer qualify, have a serious adverse 
event, or upon request of the DSMB. To withdraw from the study, subjects will 
notify Dr. Alfredo. At that time, patients will no longer take part in any 
interventions and no further research data will be collected. 
 
9. STATISTICAL CONSIDERATIONS 
 
9.1 
General Design Issues 
All analyses will be performed as intention-to-treat. Our intent-to-treat population will 
include all participants who are randomized with baseline assessment and at least one post-
randomization assessment. 
Specific Aim 1: Compare 12 weeks of slow breathing exhale>inhale vs. exhale=inhale 
on changes in physiological stress (primary outcome: heart rate variability, secondary 
outcomes: baroreflex sensitivity, catecholamines). 
 
Primary hypotheses: We hypothesize that participants with exhale>inhale vs. 
exhale=inhale will have higher increases in parasympathetic tone as measured by 
the HF HRV. 
 
Secondary hypotheses: 
3. We hypothesize that participants with exhale>inhale vs. exhale=inhale will have 
higher increases in parasympathetic tone as measured by baroreflex sensitivity 
alpha index. 
4. We hypothesize that baseline parasympathetic tone will modify response to breathing 
interventions. Specifically, we hypothesize that participants with lower parasympathetic tone 
at baseline will have the greater increases in parasympathetic tone as measured by HF HRV 
and BRS alpha index. 
 
Specific Aim 2: Compare 12 weeks of slow breathing exhale>inhale vs. exhale=inhale on changes 
in psychological stress (primary outcome- anxiety, secondary outcomes: perceived stress, arousal, 
and affect). 
 
Primary hypotheses: We hypothesize that participants with exhale>inhale vs. 
exhale=inhale will have further decreases in psychological stress as measured by the 
PROMIS Anxiety scale. 
Secondary hypotheses: 
We hypothesize that participants with exhale>inhale vs. exhale=inhale will have further decreases 
negative affect as measured by PROMIS  Depression and PANAS-X and stress as measured by 
Perceived Stress Scale, Anxiety Sensitivivity Index-3, Beck Anxiety Inventory, Body Vigilance Scale, 
and the Panic Disorder Severity Scale  
 
We hypothesize that baseline psychological stress will modify response to breathing interventions. 
Specifically, we hypothesize that participants with higher anxiety at baseline will have the further 
decreases in anxiety as measured by the PROMIS anxiety instrument. 
 
Exploratory research questions: We consider arousal as an exploratory measure in this proposal. 
In our pilot, exhale>inhale had improvements in daytime arousal vs. exhale=inhale as measured by 
the Epworth Sleepiness Scale. For the present proposal, we will examine differences in arousal from 

 
 
 
Revision Date: 10/15/18 
 
 
baseline, 6, to 12 weeks as measured by the Epworth Sleepiness Scale. We will also explore the 
relationship between baseline sleep disturbance as measured by the PROMIS Sleep Disturbance 
scale and changes in arousal. We will also explore if there are any sustained changes at 26 week in 
psychological measures and if participants continue to breathe after the intervention. 
Specific Aim 3: Compute the correlation between physiological and psychological changes (e.g. 
changes in heart rate variability correlated with changes in anxiety). 
 
Main hypotheses: We hypothesize that changes in physiological and psychological measures will 
be moderately correlated. 
 
Analyses in preparation for second phase of research project (R33): A After reaching 100% 
enrollment and baseline data collection, we will perform analyses of baseline data to identify a targeted 
population for the second phase. The purpose of these analyses is to examine the distribution of 
selected physiological and psychological stress measures in the general population. We will compare 
baseline data collected with other collected data or published studies. To identify a study population 
for R33 phase, we will choose stress thresholds (lower limit) based on the median of the general 
population or the lower quartile of previously-studied known high-stress population, whichever is 
higher.  
 
Alternative: We will investigate the effect of baseline physiological and psychological stress level on 
the intervention-induced stress change using smoothing splines with adequate knots, and identify a 
starting point of baseline stress level that is associated with desirably large change. Subjects with 
baseline stress level higher than this point will be enrolled for R33 phase study. 
 
Anticipated Results: We expect that both exhale=inhale and exhale>inhale slow breathing will 
improve physiological and psychological relaxation, and that extending the length of exhale will show 
greater benefits. 
Compared to exhale=inhale, exhale>inhale slow breathing will have greater increase in HF 
HRV and baroreflex sensitivity (Aim 1). Exhale>inhale slow breathing will also have further decrease 
in the PROMIS Anxiety instrument  (Aim 2). In addition, we expect that the changes in HF HRV will 
correlate with changes in Perceived Stress Scale in a moderate way (Aim 3). 
 
 
 
9.2 
Sample Size and Randomization 
Our pilot study demonstrates an increase in HF HRV after 6-week exhale=inhale slowing 
breathing (N=9, mean 58 ± SD 248) or after 6-week exhale>inhale slow breathing (N=9, 173 
± 531). The pooled SD from the two groups is 406, which is similar to published values 
among health adults. By 12 weeks, we expect that the difference in HF HRV between the 
two groups will be 265, and a sample size of 38 per arm will give us 80% power to detect 
this effect at a 0.05 two-sided significance level. Accounting
for 20% drop out, we will enroll 50 participants per arm. If the difference detected is 280 and 
300, the power will be 84% and 89%, respectively.   
With 50 participants per arm, we will have 80% power to detect a 5.2-point difference in 12-week 
PROMIS Anxiety Scale reduction assuming a SD of 8 and a type I error of 0.05. With a total N of 76, the 
study is able to detect a correlation coefficient of 0.7 between changes in HF HRV and changes in 
PROMIS Anxiety with a margin of error of 0.14. 
9.3 
Definition of Populations 
All analyses will be performed as intention-to-treat. Our intent-to-treat population 
will include all participants who are randomized with baseline assessment and at least 
one post-randomization assessment. 

 
 
 
Revision Date: 10/15/18 
 
 
 
9.4 
Interim Analyses and Stopping Rules 
We will not conduct interim analyses of 6 and 12-week outcomes in this clinical 
trial. After reaching 100% enrollment, baseline data will be analyzed to submit 
reports to NCCIH and prepare for second phase of grant award (R33). We will 
monitor subject accrual, adherence to interventions, and safety during the study 
period. Dr. Birdee will present data on subject accrual with research staff and IMC. 
The enrollment period will be 15 months with an anticipated enrollment rate of 7 
participants per month. 
SAE’s related to the intervention will suspend enrollment and/or the study 
intervention until a safety review is convened (either routine or ad hoc) by IMC to 
determine whether the study intervention should continue per protocol, proceed with 
caution, be further investigated, discontinued, or be modified and then proceed. If 
non-serious adverse events are related to breathing practice, then the breathing 
protocol will be modified to avoid adverse events. If the frequency of non-serious 
adverse events is higher than anticipated or alters the benefit risk ratio as determined 
by the IMC and the principal investigator, the study investigators will also modify 
the breathing protocol. Two or more specific adverse events among subjects will 
prompt modification of the breathing protocol. 
 
9.5 
Outcomes 
 
9.5.1 
Primary Outcome (Study Aim 1) 
Assessment will occur at the Vanderbilt Autonomic Dysfunction Center as per 
standardized protocol. Blood will be analyzed in the Vanderbilt University Medical 
Center laboratory according to standard procedures. Assessments will be performed 
at baseline, 6, and 12 weeks. 
Physiological stress: We will estimate autonomic tone of participants by standard 
autonomic testing of cardiovagal and sympathoneural functioning including heart 
rate variability and baroreflex sensitivity. Subjects will have fasted the night before 
testing, and testing will occur at the same time of the day for each subject to account 
for temporal variations of autonomic tone. 
 
(1) Heart rate variability: Heart rate variability determines the cardiac autonomic tone of 
the subject, measuring instantaneous heart rate fluctuations in beat-to-beat intervals during 
continuous electrocardiographic monitoring with power spectral analysis. Spectral analysis 
detects beat-to-beat values of R-R intervals21 and blood pressure values will be interpolated, 
low pass filtered (cutoff 2 Hz) and resampled at 4 Hz. Data segments of at least 300 seconds 
during 10 minutes of undisturbed recordings, will be used for spectral analysis. 
Subjects will be asked to maintain a regular breathing frequency paced to an 
auditory signal at 12 cycles per minute during recordings to control for confounding 
of respiratory sinus arrhythmia from respiration frequency. Linear trend will be 
removed and power spectral density will be estimated with the fast Fourier 
transform-based Welch algorithm using segments of 256 data points with 50% 
overlapping and Hanning window. The power in the frequency range of low 
frequencies (LF: 0.04 to <0.15 Hz), and high frequencies (HF: 0.15 to <0.40 Hz) 
will be calculated according to the Task Force recommendations.22 We will use the 
HF component of heart rate variability as an assessment of vagal control to the 
heart,23,24 and the LF component of the systolic blood pressure oscillation as an 

 
 
 
Revision Date: 10/15/18 
 
 
indicator of sympathetic modulation.25 
 

 
 
 
Revision Date: 10/15/18 
 
 
(2) 
Baroreflex sensitivity: Baroreflex sensitivity will be measured through 
changes in heart rate and blood pressure while deep breathing (6 breaths per minute) 
for one minute, Valsalva maneuver, and 10 minute standing. Testing will occur in a 
relaxing environment, with adequate time for heart rate response to normalize tests. 
Heart rate will be monitored with a 3-lead electrocardiogram (EKG) and continuous 
non-invasive finger blood pressure measurements. Baroreflex sensitivity will be 
determined based on these measurements through spectral analysis using a transfer 
function analysis. 
For heart rate response to deep breathing (E:I ratio), the patient will be asked to 
breathe deeply and slowly (about six breaths per minute) for at least one minute 
while heart rate is recorded. The ratio of maximum to minimum R-R interval will 
be calculated and compared to normative data. 
• 
For the Valsalva maneuver, subjects will be asked to breathe into a closed 
tube, generating at least 40 mm Hg of pressure for at least 10 seconds. Heart rate and 
beat-to-beat blood pressure variability (BPV) will be measured. A Valsalva ratio will 
be calculated and compared to normative data for gender and age. The blood pressure 
response will be inspected for any evidence of sympathetic dysfunction (for example, 
significant blood pressure dip during phase II of Valsalva, or failure for blood pressure 
to increase during phase IV). 
• 
For standing tests, subjects will be placed on an examination table, and then 
asked to stand-up for 10 minutes. BP and EKG recording will occur throughout the tilt. 
(3) 
Catecholamines: Plasma norepinephrine and epinephrine levels will be 
measured at baseline, 6 and 12 weeks in supine and standing positions. Catecholamine 
samples will be drawn after 20 minutes of rest with an intravenous catheter in place. 
After centrifugation, plasma will be separated and stored at -70 °C until analyzed. 
Catecholamines analyses will be performed using high-performance liquid 
chromatography with electrochemical detection.26 
9.5.2 
Secondary Outcomes  (Specific Aims 2 and 3) 
Specific Aim 2 
Psychological stress: Psychological effects related to relaxation will also be assessed at 
baseline, 6, and 12 weeks. Questionnaires will be administered online through the RedCap 
data capture system. Participants unable to access the questionnaires online will be given 
printed copies at scheduled research visits. 
 
(1) Anxiety:  
• Anxiety will be measured with the PROMIS Anxiety computerized adaptive test 
(CAT) 
(http://www.nihpromis.org/measures/domainframework1). This 29 item instrument 
assesses symptoms of self-reported fear, anxious misery, hyperarousal, and somatic 
symptoms related to arousal. The CAT allows for participant responses to guide the 
system’s choice of subsequent items from the full item bank. PROMIS Anxiety correlates 
with PANAS Negative Affect which we used in our pilot study.29 PROsetta Stone® 
provides 
a 
conversion 
of 
PANAS 
scores 
to 
PROMIS 
Anxiety 
(http://www.prosettastone.org). We used this conversation table to estimate the baseline 
and change in PROMIS Anxiety used in our statistical plan below. PROMIS Anxiety has 
been validated among health adults.  
 
We will administer other instruments of anxiety and stress as secondary measurements. 

 
 
 
Revision Date: 10/15/18 
 
 
This will provide validation of results and inform translation to other clinical populations in 
the future 
• Anxiety Sensitivity Index-3 (ASI-3) This 18-item instrument measures an 
individuals beliefs about feared consequences of symptoms associated with 
anxious arousal.1 This instrument measures three components: physical, social, 
and cognitive concerns. 
• Beck Anxiety Inventory: This 21 item instrument measures anxiety in adults2 and 
has been shown to be valid and reliable.3 
• Body Vigilance Scale: This is a validated scale to measure conscious attendance to 
internal cues and is validated in healthy and anxiety disorder adults. 4,5 
• Panic Disorder Severity Scale: This is a 7 item scale that measures panic disorder 
and agoraphobia symptom severity among adults that has shown to be valid and 
reliable.6,7 
• Perceived Stress Scale-10 (PSS): This 10 item instrument measures an individuals 
perceived stress.27,28  
 
(2) Affect: Negative affect will be measured with the PROMIS Depression CAT and 
PANAS-X. This 28 item instrument assesses negative mood, negative views of the self, 
negative social cognition, and decreased positive affect and engagement. PROMIS 
Depression has been validated among healthy adults and chronic disease populations.30,31 
The PANAS-X will be used to assess affect over the past week. This is a 60 item 
instrument that has been shown to be valid and reliable among healthy adults.8 
 
(3) Arousal: Relaxation is associated with decreased arousal and increased sleepiness. To 
account for changes in arousal we will measure daytime sleepiness with the Epworth 
Sleepiness Scale. 32 We will also administer the PROMIS Sleep Disturbance instrument 
to assess sleep quality. 
Fidelity measures: We will assess fidelity to breathing interventions through direction 
observation of the mind-body teacher, self-report, and biometric garments-HEXOSKIN. 
(1) 
Self-report: Each week, mind-body teachers observe participants performing 
the practice along with an audible metronome to measure breath rate and length of 
exhale and inhale. Mind- 
body teachers evaluate participate engagement, quality of technique, and breathe rate 
and exhale/inhale length (See Appendix for mind-body fidelity tracking form). Also 
each week, participants are asked how frequently they practiced at home by the mind-
body teacher. 
 
(2) 
Remote biometric garment: Lastly, we will use biometric garments, 
HEXOSKIN, to ascertain remote adherence to home breathing practice. As described 
previously, HEXOSKIN accurately records respiratory parameters including breathing 
rate, length of exhale/inhale through thoracic and abdominal strain gauges embedded in 
the shirt. Data for is stored in a small unit worn in the shirt. Each participant will be 
asked to wear a HEXOSKIN shirt during practice for one week for two time periods 
between weeks 3 to 8 and weeks 9 to 12. For 2-3 nights during each of these weeks the 
participant will be asked to keep the shirt on overnight to monitor sleep. Participants 
will return the shirt after one week, and data from the device will be downloaded for 
analyses. We will use VivoSense software with the Complex Respiratory Module to 
analyze breathing intervention fidelity (Vivonoetics, San Diego, CA). 
 

 
 
 
Revision Date: 10/15/18 
 
 
Specific Aim 3 
We will compute the correlation between physiological and psychological changes 
(e.g. changes in heart rate variability correlated with changes in perceived stress). 
 
9.6 
Data Analyses 
Standard graphing and screening techniques will be used to detect outliers and to 
ensure data accuracy. Distributions of continuous outcomes will be assessed for 
normality. If normality is violated, proper data transformation will be applied or non-
parametric analysis methods will be considered. Summary statistics for both 
continuous and categorical variables will be provided by randomization groups to 
describe the study sample. 
This is a two-arm randomized study with repeated measures. The effects of 
either of the slow breathing interventions on HF HRV, PROMIS Anxiety and other 
endpoints will be estimated as within-subject mean change from baseline to 12 week, 
along with their 95% confidence intervals. A paired t-test will be performed to 
compare the endpoints within the group. The difference between the two treatment 
groups will be estimated by the mean difference in the within-subject changes, along 
with their 95% confidence intervals, and a t-test will be performed. If normality is 
violated, proper data transformation will be applied. 
Mixed-effect models will also be used to analyze the data with a random 
subject effect and with intervention (exhale>inhale vs. exhale=inhale), time points 
(6 week and 12 week), baseline measurements and interaction between intervention 
and time points as fixed effects. Change in the outcomes from baseline to 6/12 
weeks will be used as responses. We will include covariates age, gender, and 
baseline psychological stress  in the models to adjust for their effects. 
Specific inferences on effects of interest will be made by reporting a point 
estimate along with a 95% confidence interval and the p-value. Hypotheses will be 
tested at the level of α=0.05. 
Based on our pilot study, we anticipate 20% missingness on post-
randomization assessment. Mixed-effects models are robust in the sense that they can 
include subjects with missing data at one time point (6 week or 12 week) to estimate 
the effects of interest. We will also perform multiple imputation for missing data to 
corroborate our findings. Specifically, all the baseline variables and outcome 
variables will be included in the imputation model, and flexible additive imputation 
models and predictive mean matching approach will be used to impute missing 
values. We will use Rubin’s method to combine the estimated regression coefficients 
and variances from the models based on ten imputed complete data sets.A Pearson 
correlation coefficient between changes in HF HRV and changes in PROMIS 
Anxiety will be calculated at each post-randomization assessment, for each 
intervention group or for all the participants respectively, along with 95% confidence 
interval. In addition, linear regression analysis of HF HRV changes on PROMIS 
Anxiety changes, age and gender will be conducted. 
Per-protocol analysis will be also performed as a sensitivity analysis, 
and only participants who practice breathing at home at least 3 times a week 
and achieve prescribed breathing rate and ratio for at least 2 time periods after 
randomization will be included. 
10. DATA COLLECTION AND QUALITY ASSURANCE 
 
10.1 
Data Collection Forms 

 
 
 
Revision Date: 10/15/18 
 
 
Research staff blinded to treatment assignment will administer a laptop or 
tablet to collect data through REDCap. Subjects may opt to complete 
questionnaires online at home. Physiological measures will be assessed by a 
blinded lab technician blinded at the Autonomic Dysfunction Center with results 
entered into REDCap. Physiological measures will be performed by research staff 
and documented through data software programs with migration to REDCap. 
To protect confidentiality, all data will be stored in a locked file cabinet 
located in the secured office of the research coordinator. All computers and data 
files will be password protected. All data processed will be in aggregate form and 
data collection forms will only be labeled with subject’s unique identification 
number. 
Data collection forms are presented in the appendices as outline in the 
Table of Contents. 
 
 
10.2 
Data Management 
The principal investigator will be responsible for data management. 
 
10.3 
Quality Assurance 
 
10.3.1 Training 
All research staff will have completed the online Collaborative Institutional 
Training Initiative Training (CITI) prior to participation in research activities. 
Staff will participate in ongoing monthly workshops offered by the Vanderbilt 
Human Subjects Protection Program Responsible Conduct of Research-related 
principles and practices offered through the Human Research Protection Program. 
Dr. Birdee will directly oversee training of research staff in regards to conducting 
to the planned research. 
 
10.3.2 Metrics 
Dr. Birdee will monitor data collection to assure quality control. Data will be 
entered into REDCAP, which is a web-based application to support data capture 
for research studies, allowing assessment of data collection quality including 
timely and complete data entry. Dr. Birdee will provide monthly updates to the 
IMC and research mentors regarding patient recruitment and enrollment. 
 
10.3.3 Protocol Deviations 
Protocol deviations will be captured through direct observation by research 
staff regarding patient recruitment and enrollment, intervention administration, 
adherence, and safety monitoring. Protocol deviations will be reviewed by the 
principal investigator and discussed with research mentors and as needed with the 
IMC. 
 
10.3.4 Monitoring 
Dr. Birdee will monitor for protocol compliance, data quality, and review of 
documentation to assure protocol compliance. This will include review of 
inclusion/exclusion criteria to insure eligibility (during recruitment), informed 
consents (upon enrollment, adverse event reporting (upon occurrence), and data 

 
 
 
Revision Date: 10/15/18 
 
 
collection in REDCap (monthly basis). 
 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY 
 
11.1 
Institutional Review Board (IRB) Review 
This protocol and the informed consent documents and any subsequent 
modifications will be reviewed and approved by the IRB or ethics committee 
responsible for oversight of the study. 
11.2 
Informed Consent Forms 
A signed consent form will be obtained from each participant. For participants who cannot 
consent for themselves, such as those with a legal guardian (e.g. person with power of 
attorney), this individual must sign the consent form. The consent form will describe the 
purpose of the study, the procedures to be followed, and the risks and benefits of 
participation. A copy will be given to each participant or legal guardian and this fact will be 
documented in the participant’s record. 
 
11.3 
Participant Confidentiality 
Any data, specimens, forms, reports, video recordings, and other records that leave the 
site will be identified only by a participant identification number (Participant ID, PID) to 
maintain confidentiality. All records will be kept in a locked file cabinet. All computer 
entry and networking programs will be done using PIDs only. Information will not be 
released without written permission of the participant, except as necessary for monitoring 
by IRB, the FDA, the NCCIH, and the OHRP. 
 
11.4 
Study Discontinuation 
The study may be discontinued at any time by the IRB, the NCCIH, the OHRP, the 
FDA, or other government agencies as part of their duties to ensure that research 
participants are protected. 
 
 

 
 
 
Revision Date: 10/15/18 
 
 
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and initial validation of the Anxiety Sensitivity Index-3. Psychol Assess. Jun 2007;19(2):176-
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Fydrich T, Dowdall D, Chambless DL. Reliability and validity of the Beck Anxiety Inventory. 
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Schmidt NB, Lerew DR, Trakowski JH. Body vigilance in panic disorder: evaluating attention 
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Shear MK, Rucci P, Williams J, et al. Reliability and validity of the Panic Disorder Severity 
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Watson D, & Clark, L. A. The PANAS-X: Manual for the positive and negative affect schedule: 
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