A Randomized Control Trial Treating Depression With Yoga and Coherent Breathing Versus Walking in Veterans
Phase: NA
Conditions: Depressive Disorder, Major, Post Traumatic Stress Disorder
Sponsor: VA Office of Research and Development

A Randomized Control Trial Treating Depression With Yoga and Coherent Breathing Versus Walking in Veterans 
NCT03489122 
Document Date: 3-07-2022 

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 HUMAN SUBJECTS RESEARCH 
PROTOCOL 
 
 
Project Title: A Randomized Controlled Trial Treating Depression with Yoga and Coherent 
Breathing Versus Walking in Veterans 
 
Short Title: RCT Treating Depression with Yoga or Walking in Veterans 
Protocol Version and Date: Version 5 December 31, 2021 
Principal Investigator (PI): Chris C. Streeter, M.D. 
 
Co-Investigators: Chun Zuo, Ph.D., Principal Investigator for the McLean Hospital. 
 
Institution(s): The VA Bedford Healthcare System (VABHCS) is the coordinating site. The McLean 
Hospital, Belmont MA will be the site for MRI imaging. 
 
1.0 
OBJECTIVE AND SPECIFIC AIMS: 
 
Aim 1: To determine in Veterans, the effectiveness of an RCT of 12-weeks of the yoga intervention 
versus walking intervention, with depression and mood assessed at baseline prior to randomization, 
and prior to and immediately following yoga or walking interventions at weeks 4, 8, and 12, and before 
each scan. 
Hypothesis I: In this working clinical hypothesis, subjects in the yoga group will exhibit greater 
improvements in depressive symptoms and mood ratings over the study compared to the walking 
group. 
 
Aim 2: To measure changes in thalamic GABA levels at Scans 1, 2, and 3. (3) 
Hypothesis II: In this working translational hypothesis, GABA levels will increase and depressive 
symptoms will decrease over the course of the 12-week intervention in the yoga group compared to the 
walking group. 
 
Aim 3: To examine changes in RSA acquired at baseline prior to randomization, and prior to and 
immediately following yoga or walking interventions at weeks 4, 8, and 12, and before each scan. 
Hypothesis III: In this working translational hypothesis, RSA levels will increase and depressive 
symptoms will decrease over the course of the 12-week intervention in the yoga group compared to the 
walking group. 
 
Exploratory Aim: If an individual has symptoms of Post Traumatic Stress Disorder (PTSD) at 
screening, there will be a greater decline in PTSD symptoms with treatment in the yoga compared to 
the walking group. The use of the 3T scanner allows the collection of pre and post exploratory data for 
cortical thickness, resting state, and executive function. 
 
2.0 
BACKGROUND AND SIGNIFICANCE 
 
2.1 
Background Cost of Major Depressive Disorder (MDD): The World Health Organization Global 
Burden of Disease study estimated that depression is the single most burdensome illness during the middle 
years of life, in both developing and developed countries, with no other disease accounting for even half of 
the total burden.(1) Advances in the treatment of MDD include antidepressants, psychotherapy, and somatic 
treatments such as Electroconvulsive Therapy (ECT), Vagal Nerve Stimulation (VNS) and Transcranial 
Magnetic Stimulation (TMS). Although these treatments have been found effective for reducing symptoms of 

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MDD in RCTs, 40 to 50% of individuals with MDD do not achieve remission.(2,3) Even the Sequenced 

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Treatment Alternatives to Relieve Depression (STAR*D) study of non-psychotic MDD subjects, which has 4 
treatment steps for advancing individuals who fail medication trials, has an overall cumulative remission rate 
of only 67%.(2, 4) Notably, individuals who have one depressive episode are at increased risk for another 
depressive episode. However the risk of recurrence is significantly lower in 61% of individuals with 
asymptomatic recovery from a Major Depressive Episode, and they remain free of a relapse or recurrence 
4.2 times longer than those who continue to exhibit subsyndromal depressive symptoms.(3) The use of 
yoga-based therapies as monotherapy or adjunctive treatment for depression represents a promising area 
of research to address the current short falls in available therapies. 
 
2.1.a. Vagal-GABA Theory [Figure 1]: The Vagal-GABA theory developed by our group, explains why 
yoga practices should benefit numerous comorbid disorders that are common in the VA population, by 
increasing activity in parasympathetic and GABA systems. It is hypothesized that stress induces: 1) 
imbalance of the ANS, with decreased parasympathetic nervous system (PNS) and increased 
sympathetic nervous system activity (SNS), 2) an underactive GABA system, and 3) increased 
depressive symptoms. It is further hypothesized that yoga and coherent breathing correct underactive 
PNS and GABA systems in part through stimulation of vagal nerves, reducing allostatic load caused by 
stress and decreasing depressive symptoms. 
 
2.1.b. How Yoga Affects the Autonomic Nervous System: According to the Polyvagal Theory 
developed by Porges (1995), the myelinated vagus, the most evolutionarily advanced component of the 
ANS, supports social engagement, engenders feelings of safety, and promotes calm states consistent 
with the metabolic demands of growth, repair, and restoration.(5) Respiratory Sinus Arrhythmia (RSA) 
is a validated measure of myelinated vagal efferent out flow from the nucleus ambiguous to the sino- 
atrial node.(5) RSA represents only the myelinated vagal contribution to cardiac vagal tone, as 
indicated by the high frequency (HF) component of heart rate variability (HF-HRV). Increases in RSA 
reflect increases in parasympathetic activity and vagal dominance.(5) The proposed study will measure 
RSA because it is specific to the myelinated vagal contributions to changes in heart rate, thus excluding 
non-vagal contributions to changes in HRV.(5) The sequence of evolutionary development ascends 
from the unmyelinated vagus to the 
SNS and, most recently 
inmammals only, the myelinated 
vagus. The myelinated vagus 
conducts impulses more rapidly 
than the SNS and the 
unmyelinated vagus. Myelinated 
vagal efferent fibers from the 
nucleus ambiguous serve as the 
vagal brake, enabling rapid 
control of heart rate by increasing 
vagal tone (stepping on the brake) 
to reduce heart rate or decreasing 
vagal tone (letting up on the 
brake) to accelerate heart rate. 
The vagal nerve, Cranial Nerve X, 
is the primary parasympathetic 
pathway of the peripheral nervous 
system. Inspiration and expiration, 

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through stretch receptors in the alveolar walls and other respiratory structures (e.g. diaphragm, thoracic 
musculature), send information to the brain via the vagus nerve. The normal adult respiratory rate is 
about 12 to 20 breaths per minute. Studies of yoga breathing exercises using slow respiratory rates 
have demonstrated reduction in sympathetic activity along with increased parasympathetic activity, 
often measured by increased HF-HRV.(6-8) Vagal afferents in the lungs send information to the 
nucleus tractus solitarius, the primary input nuclei for the vagus in the medulla; through a series of 
relays, this leads to the transmission of information through myelinated vagal efferents from the nucleus 
ambiguous to the sino-atrial node, the cardiac pacemaker (See Figure 1).(9) In healthy controls, Iyengar 
practitioners, depressed women, and individuals with low back pain, interventions composed primarily 
of yoga postures are associated with increased HRV. Thus, HRV has become an established biomarker 
for measuring effects of yoga postures.(10-12) Systematic reviews found risk of bias using the 
Cochrane Criteria in studies assessing HRV in RCTs comparing yoga with a control intervention.(13) 
Due to the methodological weakness in the study designs, the review concluded that more research 
was needed concerning the effect of yoga on HRV. This proposed study fills that gap because it is 
designed to comply with the Cochrane Collaboration Tool for RCTs.(14) In a study of depressed women 
assigned to a 12-week Hatha yoga intervention or an untreated control group, there was a significant 
decrease in Beck Depression Inventory II (BDI-II) scores and a significant increase in HF-HRV.(12) 
Yoga stretching is considered a form of self-massage, which has been associated with increased HF- 
HRV and decreased Spielberg State Trait Anxiety Inventory (STAI) scores.(15) The use of RSA, a 
component of HRV, as a biologic marker for the effect of yoga breathing and postures in this proposal is 
supported by the above studies that used the BDI-II and STAI with HRV, the same dependent variables 
used in our dosing study and proposed in this study. Slow breathing exercises have also been linked 
with improved mood and increased HRV.(16) Subjects with MDD who were treated with biofeedback 
that used breathing at a resonance frequency of 4.5 to 6.5 breaths per minute, demonstrated increased 
HRV and improved mood.(8) The results of these studies support the use of RSA as a biomarker for 
both yoga postures and breathing. The Neurovisceral Integration Model of Thayer (2000) suggests that 
ANS imbalance, with an underactive PNS, may be the final common pathway between negative 
emotions and poor health (e.g., depression, PTSD, anxiety, panic), as well as medical conditions (e.g., 
cardiovascular disease).(17-19) In disorders with stress-induced ANS imbalance, optimizing ANS 
imbalance entails increasing PNS under activity and/or reducing SNS over activity. Increased HF-HRV, 
a marker for increased PNS activity, indicates that the stress response system has greater flexibility to 
respond to challenges.(16) Stress is associated with inflammation and immune imbalance, both of 
which may be ameliorated by yoga stretching. The connective tissue network is an integrated, whole- 
body system that is amenable to physical manipulation by massage, yoga, qigong, and tai chi.(20) 
Fasciae are composed of an extracellular connective tissue matrix that forms structures surrounding 
every organ of the body, integrates the musculoskeletal system, and houses the blood and lymphatic 
vasculature.(20) Stiffness and lack of mobility of fascia has implications beyond the inability to move 
adequately. An extensive literature links physical mobility and health, including mental health.(20) 
Stretching of connective tissues may have local anti-inflammatory effects independent of vascular, 
neural, or other systemic factors.(21) Exercises with a prominent stretching component (e.g. yoga) 
have been found to decrease levels of circulating pro-inflammatory cytokines. In comparison to expert 
hatha yoga practitioners, the novices’ serum IL-6 levels were 41% higher, and the odds of a novice 
having detectable C-Reactive Protein were 4.75 times as high as that of an expert.(22) In a 6-month 
RCT, subjects with inflammatory bowel disease (IBD) were randomized to coherent breathing plus 
qigong movement intervention or an educational control group. The coherent breathing group 
demonstrated significantly decreased stress, improved mood, reduced IBD symptoms and decreased 
C-Reactive Protein versus no changes in the control group.(23) That study is the first to show that in 
patients with IBD (a stress-driven disorder of immune response), coherent breathing and qigong (a 
Chinese form of yoga) is associated with reduced inflammation (presumably via vagal anti- 
inflammatory pathways) and deceased stress-related immune imbalances.(23) A meta-analysis of more 
than 300 empirical articles describes the relationship between psychological stress and parameters of 

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the immune system in humans.(24) Chronic stress alters immune responses. A change in the immune 
system from flexible and balanced to inflexible and unbalanced may contribute to increased 
vulnerability to stress-related immune dysregulation. An additional mechanism whereby yoga postures 
and coherent breathing alleviate depression may be through counteracting inflammatory abnormalities 
seen in some depressed subjects.(9) 
 
2.1.c. Yoga Treatments in Disorders with Low Parasympathetic and GABA Activity: MDD, PTSD, 
Obsessive-Compulsive Disorder, Generalized Anxiety Disorder and epilepsy, are associated with low 
parasympathetic and GABA activity. Interventions that increase parasympathetic activity (measured by 
HF-HRV or RSA) or increase GABA activity (measured by MRS) are associated with clinical 
improvements in a number of disorders of clinical relevance to Veterans.(25-36) These disorders 
demonstrate symptom reduction when treated with pharmacologic agents that increase GABA activity, 
and when treated with yoga in RCTs.(37-43) Iyengar yoga has been shown to increase 
parasympathetic tone and has been successfully used to treat depressive symptoms in controlled 
studies.(11, 44, 45) Further, studies have found yoga to be effective as a monotherapy and as 
adjunctive therapy for individuals with MDD being treated with antidepressants, but these were not 
RCTs.(44, 46) Although there is substantial evidence to support the use of yoga in treatment of MDD, a 
recently published review found yoga to have only Level 3 evidence (Level 1 was highest, Level 4 was 
lowest), as an adjunctive treatment for depression due to insufficient safety data and non-randomization 
of studies.(47) Our recently published dosing paper, recently submitted safety paper and the current 
proposal address these gaps by using the Consolidated Standard of Reporting Trials (COHORT) and 
the Cochrane Collaboration Tool for assessing risk of bias in randomized trials and by careful 
monitoring of adverse events for safety data.(14, 48) 
 
2.1.d. Trauma Response Spectrum: The Trauma Spectrum Response includes emotional 
dysfunction, (e.g., depression, anxiety, PTSD and anger), cognitive dysfunction, (e.g., decreased 
attention, concentration, memory and problem solving), somatic dysfunction, (e.g., fatigue and sleep 
disturbances), and alcohol and substance use disorders.(49) It is observed that disorders within the 
Trauma Response Spectrum are co-morbid and share fundamental imbalances – i.e., under activity in 
the PNS and GABA systems and over activity in the SNS. These observations support the concept that 
comorbid disorders observed in the Veteran population, such as MDD, PTSD and Alcohol Use Disorder 
(AUD) are based on the same transdiagnostic biological abnormalities. The use of an intervention that 
addresses abnormalities in ANS balance is novel, provides a new entry point for treatment and may 
enhance available treatments. In an RCT of 30 Australian Vietnam veterans, diagnosed as 100% 
disabled due to PTSD by the Australian VA and who had failed 30 years of treatment trials, including 
multiple medication combinations, a 5-day multi-component yoga and breathing intervention resulted in 
significant recovery from PTSD and depression at week-6 compared to the control group, and showed 
further improvements at 6 months.(50) On the Clinician Administered PTSD Scale, the effect size from 
baseline to week-6 was high at 0.90 and 2.9 at 6 months. Scores on the Center for Epidemiological 
Studies Depression Scale (CES-D) and Post-traumatic Stress Disorder Checklist Military (PCLM-17) 
also decreased significantly from baseline to week-6 and month-6. A similar study in 21 US Veterans of 
the wars in Iraq and Afghanistan found significant recovery in PTSD, anxiety, and physiologic measures 
of hyperarousal.(51) The proposed study, by monitoring PTSD symptoms in MDD subjects, will provide 
information on whether changes in symptoms of MDD and PTSD in response to the proposed 
intervention are correlated in this population, an observation that would support the transdiagnostic 
nature of these conditions, and the potential of an intervention that addresses a common and 
fundamental imbalance, having a far reaching impact on the care of Veterans.(49, 52) 
 
2.2. 
Preliminary Data 
 
2.2.a. Summary of Work Done: Over the last decade, our group has completed four studies, with 

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translational and clinical components, demonstrating that yoga interventions are associated with 
increased thalamic GABA, increased RSA, improved mood, and decreased anxiety. These studies 
included experienced yoga practitioners, healthy controls, yoga naïve healthy controls, subjects with 
MDD and low back pain, subjects with MDD and PTSD, and subjects with MDD.(9, 53, 54) Previous 
studies conducted in civilians were supported by federally funded grants (R21, R01, K03), and resulted 
in peer reviewed publications, and manuscripts under review and in preparation. 
 
2.2.b. Study 1 (54), Streeter et al 2007, a cross-sectional study, compared changes in GABA levels using 
magnetic resonance spectroscopy (MRS) in experienced yoga practitioners and healthy controls with 
no yoga experience at baseline (Scan 1), followed by 60-minutes of hatha yoga practice in yoga 
practitioners or reading magazines in healthy controls. This was immediately followed by Scan 2. 
GABA-to-Creatine ratios (GABA/Cr), measured in a 2-cm axial brain slab, showed 27% increase in 
GABA/Cr in yoga practitioners after the yoga session, but no significant change in healthy controls (t = - 
2.99, df = 7.87, p = 0.018). This was the first study to document a significant increase in GABA 
associated with a yoga intervention. 
 
2.2.c. Study 2 (53), Streeter et al 2010 addressed the possibility that increased GABA/Cr in Study 1 could be 
due to exercise. In a longitudinal RCT, eligible subjects who were yoga naïve, with no Axis 1 disorders, 
no use of medications known to influence GABA receptors, and no current yoga or mind-body practices 
received a baseline scan (Scan 1). Subjects were then randomized to a 12-week Iyengar yoga 
intervention or a group walking intervention that was matched to the yoga intervention for (1) Metabolic 
Equivalents (METS) by walking 2.5 miles an hour on a flat surface, (2) interaction with research staff 
and (3) group effect.(55) After the 12-week intervention, subjects underwent Scan 2, immediately 
followed by a 60-minute yoga or walking intervention, depending on group assignment, prior to Scan 3. 
 
2.2.d. Changes in Mood Scores: Study 2 utilized the Spielberg State Trait Anxiety Inventory (STAI), 
State and Trait subscales, and the Exercise Induced Feeling Inventory (EIFI) with subscales for 
Revitalization, Tranquility, Revitalization, and Physical Exhaustion.(56, 57) These scales were selected 
because they can be given repeatedly to measure current mood states over time. Mood assessments 
were obtained at baseline, before each scan, and at weeks 4, 8, and 12. Groups did not differ on 
demographic variables or STAI Trait anxiety scores. Tonic changes over the course of the study (Scan 
2 – Scan 1) for mean mood scores showed significant increases in the yoga group for Revitalization 
(1.8 ± 2.5, t18 = 3.21, p = 0.005). Acute changes from pre- to post-intervention (Scan 3 – Scan 2) for 
mean mood scores showed significant increases in the yoga group for Revitalization (2.5 ± 2.7, t18 = 
4.12, p < 0.001) and Tranquility scores (2.0 ± 1.8, t18 = 4.77, p < 0.001), and a significant decrease in 
STAI-State anxiety scores, consistent with decreased anxiety (-5.2 ± 5.5, t17 = -4.05, p < 0.001). A 
Generalized Estimating Equation (GEE) analysis of mean mood scales at baseline, and weeks 4, 8, 
and 12, showed the yoga group to have significantly greater improvement than the walking group on all 
subscales of the EIFI, with significant increases in Tranquility, Revitalization and Positive Engagement, 
and significant decreases in Physical Exhaustion. 
 
2.2.e. Study 3 (9), Streeter et al 2012: This study compared GABA/Cr in subjects with MDD who were 
enrolled in a 12-week trial of Hatha yoga for low back pain (Saper 2009) and with healthy controls from 
Study 2.(58) Iyengar yoga is a subset of Hatha yoga. The Iyengar yoga 12-week intervention from 
Study 2 and the Hatha yoga 12-week intervention for Low Back Pain were reviewed by a certified 
Iyengar instructor and found comparable. The MRS protocols used to collect GABA/Cr were identical in 
both studies. This allowed Scan 1, obtained before the 12 week interventions and Scan 2 after the 
interventions to be compared in healthy controls (n=19) from Study 2 and subjects with MDD and low 
back pain from Study 3 (n=2). At baseline, the MDD subjects with low back pain demonstrated lower 
GABA/Cr, which increased over the course of the 12-week yoga intervention, as depressive symptoms 
and low back pain declined. 

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2.2.f. Acceptability and Feasibility of Yoga to Veterans (59): A RCT of the yoga for low back pain 
intervention discussed in C.3.a versus an educational control was conducted in Veterans at the 
VABHCS, under the direction of Dr. R. Saper, co-investigator of Study 3: 283 Veterans were 
interested, 210 completed telephone screening, 28 (13%) were ineligible, 182 (87%) met eligibility, 131 
(62%) completed in person consent, 120 (57%) were randomized to yoga for low back pain or 
educational intervention. Randomization was completed in 10 months with follow up for 24 months. 
Over 90% were male, 49% were Non-Hispanic white. Available data show that out of the 120 
randomized subjects, 57 have completed the intervention, 39 are schedule to complete the intervention 
in March, and 24 are scheduled to complete the intervention in June. These data demonstrate that a 
yoga protocol similar to the proposed yoga intervention is acceptable to Veterans at the VABHCS, the 
site of the proposed study. 
 
2.2.g. Study 4 Streeter et, al 2017: Study 4 is a dosing study that filled a gap in the literature, as there were no 
published studies on the optimal dose of a hatha yoga intervention for MDD. Study 4 refined the Study 
2 Iyengar yoga protocol by adding a 20-minute coherent breathing exercise at five breaths per minute 
to further increase PNS activation, thereby maximizing the effect of the intervention. This increased the 
intervention to 90-minutes, ~60-minutes of postures, transitions and 20-minutes of coherent breathing. 
The dosing study was a longitudinal RCT wherein subjects with MDD were randomized to 12 weeks in 
a high dose group (HDG) of three 90-minute yoga classes per week and four 30-minute homework 
sessions, or a low dose group (LDG) of two 90-minute yoga classes per week and three 30-minute 
homeworks. Eligible subjects, 18-64 years old with MDD, had baseline Beck Depression Inventory II 
(BDI-II) scores ≥14 (with higher scores representing greater depressive symptom severity) and were 
not on antidepressants, or were on a stable dose of antidepressants for ≥3 months. Exclusion criteria 
included current psychotherapy for depression, history of psychosis or bipolar disorder, active alcohol 
or substance abuse, history of suicidal ideation with intent within the last year (suicidal ideation without 
intent was allowed), current mind-body practices, nicotine use, more than 5 drinks on three occasions 
in the last 3 months, and medications directly affecting GABA receptors (e.g., sedatives 
benzodiazepines, antiseizure medications). Anxiety Disorders were allowable and resulted in the 
enrollment of the following: 8 PTSD current, 1 PTSD in full remission, 4 PTSD sub-threshold, 3 Panic 
Disorder in partial remission, 1 Panic Disorder in 
full remission, and 1 Social Phobia current. 
 
2.2.h. Depressive Symptoms, Compliance 
and Safety (Figure 2): Retention was excellent, 
with 93% (30/32) of randomized subjects 
completing the study: HDG (n = 15), and LDG 
(n=15). Compliance was excellent for classes, 
homework and total yoga minutes. Compliance 
for total yoga minutes was 87 ± 28% for the 
HDG and 84 ± 19% for the LDG, with no 
difference in compliance between groups (p = 
0.77). This resulted in a significant difference 
between groups in total yoga minutes with 4,075 
± 1,314 minutes for the HDG and 2,737 ± 625 
minutes for the LDG (p = 0.001). There were no serious adverse events related to the study. To our 
knowledge, this is the first study to report significant yoga dosing differences between groups and 
demonstrates our group’s ability to recruit and retain subjects who are compliant with the proposed 
yoga protocol. Demographic variables and BDI-II scores at screening did not differ significantly between 
HDG and LDG. BDI-II scores declined significantly from screening through week 12 for the HDG (-18.6 
± 6.6, p<0.001) and the LDG (-17.7 ± 9.3, p<0.001). As there was no difference between groups in BDI- 

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II scores, a two-sample paired t-test conducted for the full cohort at baseline (26.2 ± 7.4) compared to 
week-12 (8.0 ± 6.4), demonstrated a significant reduction in depressive symptoms (t29 = -12.5, p < 
0.001). A Generalized Estimating Equation (GEE) analysis at baseline, 4, 8, and 12 weeks with BDI-II 
scores as the dependent variable and Cumulative Yoga Minutes as the independent variable was 
statistically significant (β = -0.002, z = -2.31, p = 0.02), with depressive symptoms declining as yoga 
minutes increased (See Figure 2). 
 
2.2.i. Depressive Symptoms and Mood Scales (Table 1): Using a two-sided Fisher’s exact test 
comparison, there were no significant differences observed between groups from baseline through 
week-12 in either the proportion of responders, > 50% decrease in BDI-II (p=0.65) in 87% of HDG 
(13/15) and 73% of LDG (11/15), or the proportion of remitters, BDI-II scores ≤14 at week 12 (p = 1.00) 
in 93% of HDG (14/15) and in 87% of LDG (13/15). Statistically significant differences occurred in the 
proportion of subjects with BDI-II scores ≤10 at week-12 (p = 0.04), 93% of HDG (14/15) and 53% of 
LDG (8/15). GEE analysis of mood scales at baseline and weeks 4, 8 and 12 for the full cohort showed 
significant improvements in mood and decreases in anxiety when corrected for multiple comparisons 
(Table 1). The Pittsburg Sleep Questionnaire Inventory (n=30) showed significant improvement in sleep 
from baseline (7.8 ± 3.9) to 12-weeks (5.3 ± 3.6) (t29 = -4.91, p < 0.001). Given that the HDG and LDG 
did not differ for BDI-II scores ≤ 14 at week-12, considering the time demands of a tri-weekly 
intervention, and the significant results in the Study 2 with two yoga interventions per week, the 
proposed study will use the LDG model, two yoga interventions per week. 
 
2.2.j. Thalamic GABA/Cr (Figure 3): Figure 3 shows a representative proton spectrum from a LDG 
subject at baseline (Scan 1), after 12-weeks of yoga and breathing intervention (Scan 2), and 
immediately after a 90-minute yoga and breathing session at week 12 (Scan 3). There is a noticeable 
increase in the GABA peak from Scan 1 (blue spectrum) to Scan 2 (yellow spectrum) to Scan 3 (red 
spectrum). Because the proposed study will use two yoga classes per week, a one-sample t-test 
comparing the change in GABA/Cr from Scan 1 (pre randomization) to Scan 3 (post 12-week 
intervention) in the LDG (that also used two classes) shows a statistically significant increase in 
thalamic GABA/Cr (t14 = 2.51, p = 0.02). 
 
2.2.k. Reanalysis of Study 2 Data: In Study 2, healthy controls participated in a yoga intervention and 
study design similar to Study 4, with Scan 1 at baseline, Scan 2 after a 12-week yoga intervention and 
Scan 3 immediately after a yoga intervention. In both Studies 2 and 4, GABA is reported as GABA/Cr 
ratios, which is standard in the field of MRS. In order to compare values from Study 2, collected in 
analog format on the 4T scanner, with values from Study 4, collected in digital format following a 
system upgrade to the same 4T scanner, Study 2 data were converted to digital format and re-analyzed 
using the same spectral fitting algorithms as in Study 4. Consistent statistical conclusions were 
maintained. 

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2.2.l. Comparison of Thalamic GABA/Cr in Healthy 
Controls and Subjects with MDD (Figure 4): A fixed 
effects, omnibus one-way ANOVA, was used to detect 
differences in GABA/Cr in subjects with MDD (LDG, 
n=15) from Study 4 and healthy controls (n = 18) from the 
yoga group in Study 2. This analysis revealed significantly 
higher GABA/Cr in healthy controls at Scan 1 (F1,32 = 
6.486, p = 0.016) compared to MDD subjects. Differences 
between these groups were no longer significant at Scan 
2 (F1,32=2.636, p = 0.115) or Scan 3 (F1,32 = 1.695, 
p=0.203), as GABA/Cr of subjects with MDD increased 
(normalized) to the level of non-depressed healthy 
controls over the 12-week intervention. 
 
2.2.m. Parasympathetic tone measured with 
Respiratory Sinus Arrhythmia (RSA) (Figure 5): In 
Study 4, RSA values were derived from an 
electrocardiogram (ECG) recording over a three stage 
protocol: 1) a 5-minute rest, 2) a 3-minute step test that 
removes the vagal break, and 3) a 5-minute recovery 
period. RSA was measured at baseline and after an 
intervention at weeks 4, 8, and 12. No differences 
between the HDG and LDG were observed for baseline or 
recovery RSA. A GEE analysis of changes over the 12- 
week intervention for the full cohort showed no RSA 
differences at rest (β = -0.01, z = -0.74, p = 0.46), but an 
increase in RSA during recovery (β = 0.05, z = 2.12, p = 
0.03), consistent with the recovery stage being the best 
marker of tonic changes from the beginning to the end of 
the study. Midway through the study, an additional RSA 
was added pre the week-12 intervention, permitting 
comparison of pre- and post- week-12 intervention RSA 
values (n=16). Rest stage (F1,15 = 11.06, p = 0.005) and 
recovery stage changes (F1,15 = 3.82, p = 0.07) were 
significant from pre- to post- 12-week intervention. The 
acute changes in both rest and recovery stages observed 
from pre- to post-intervention, support the hypothesis that 
yoga acutely increases resilience measured by increased 
RSA in the rest and recovery stages. Tonic changes were 
only significant in the recovery stage, suggesting that the 
recovery stage is more sensitive to resilience. 
 
2.2.n. Correlation of Thalamic GABA/Cr and RSA: 
Because the proposed study will use two yoga classes 
per week, correlational analysis of GABA and RSA was 
conducted on data from the LDG. Acute changes in 
GABA were measured at the end of the 12-week 
intervention from pre-yoga intervention (Scan 2) to post- 
yoga intervention (Scan 3). At the end of the 12-week 
yoga intervention, acute RSA changes were also 

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measured before and immediately after a yoga intervention. A Spearman rho correlation (two-tailed) of 
the difference between this 12 week pre- and post- intervention recovery RSA for Scan 3 – Scan 2 
GABA/Cr was statistically significant (rho7 = .821, p = 0.023). This supports the hypothesis that RSA is 
significantly correlated with GABA. 
 
2.2.o. Safety and Reduced Suicidal Ideation under review Streeter: In Study 4, mild side effects related to 
the study occurred, most commonly muscle soreness (n=13), which resolved over the course of the 
study. There were no serious Adverse Events related to the study. No subjects developed suicidal 
ideation during the study; 9/10 subjects reporting suicidal ideation without intent at screening on the C- 
SSR, with 8/9 reporting the resolution of this symptom over the course of the study. This preliminary 
evidence suggests that yoga and coherent breathing are safe and effective in the treatment of MDD, 
and may reduce suicidal ideation. 
 
2.2.p. Yoga as an Adjunctive to Pharmacologic Treatment in Study 3 and 4: Approximately 40% of 
individuals treated with pharmacology for MDD do not achieve full remission, which places them at 
increased risk for recurrence and relapse.(3) Study 3 (n = 2) and Study 4 (n = 2) include subjects on 
stable doses of antidepressants for greater than three months who still met criteria for MDD when 
enrolled in the 12-week yoga protocol. Subjects were on the following medications that act on the 
monoamine system: duloxetine and venlafaxine, serotonin-norepinephrine reuptake inhibitors (SNRI); 
fluoxetine, a serotonin selective reuptake inhibitor (SSRI); and bupropion, a norepinephrine-dopamine 
reuptake inhibitor (SNDI). All subjects exhibited decreased MDD symptoms with addition of the 12- 
week yoga intervention, as measured by PHQ-9 in Study 2 and BDI-II in Study 4. These observations 
support the hypotheses that yoga postures and breathing interventions corrected an imbalance of 
sympathetic over activity and parasympathetic under activity, at the level of the vagal nuclei in the 
medulla. The proposed yoga intervention shows promise as augmentation to pharmacotherapy that 
addresses imbalances in the monoamine systems found at higher levels of the neural axis in the pons 
and midbrain. 
 
2.2.q. How would the results of this study impact clinical practice and/or plans for future 
studies? There is evidence that Complementary and Alternative Medicine (CAM) among Veterans may 
be greater than in the general population, with 50% of the Veterans in the Southern Arizona VA medical 
Facilities reporting CAM use compared to 36% to 38% of the general US population.(60) In veterans 
with chronic pain, up to 76% of those not currently using CAM therapies reported wanting to use CAM 
was if available in their VA facility.(61) A large number of VA facilities provide some type of CAM.(60) 
There is increasing recognition that multimodal treatments are needed to address issues of depression, 
anxiety and pain in the VA population. A Mindfulness Based Stress Reduction (MBSR) study in 
Veterans reported decreased anxiety, depression and suicidal ideation.(60) While this study 
demonstrates the potential benefit and acceptability of MBSR that included gentle yoga, this was a self- 
referred open-label study. More RCTs are needed to increase the evidence base such that these types 
of treatment can be integrated into multimodal treatment plans for Veterans. Suicides in the Veteran 
population have become an urgent public health concern. Historically lower than civilian suicide rates, 
in the past decade, suicide rates in the Veteran population have doubled, surpassing death due to 
being killed in action.(62) The risk of suicide in Veterans with diagnoses of MDD, PTSD, and Alcohol or 
Substance Use Disorder also is increased. There are few evidenced based therapies addressing 
suicidality.(63) The proposed intervention has demonstrated effectiveness in reducing suicidal ideation. 
Over a 12-week trial in MDD, the proposed intervention documented, both response and remission of 
MDD symptoms, over 80% compliance, mild side effects, and no increases in depressive 
symptoms.(64) The proposed intervention was effective in reducing depressive symptoms in subjects 
who were medication free and in subjects who were still depressed on stable pharmacologic treatment 
for at least 3 months. Therefore, the proposed trial has potential to support a significant vertical change 
in the effectiveness of care provided to Veterans suffering from MDD, as monotherapy or as an adjunct 

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to existing treatments. This is not a treatment resistant depression study, broadly defined as failure on 
trials with at least two pharmacologic agents for an adequate period of time at an adequate dose. This 
study addresses the need of the 40% of individuals being treated for MDD, who do not achieve 
asymptomatic remission. The proposed research will employ established Iyengar trained teachers to 
insure the safety, quality and consistency of the intervention in this efficacy study. Generalizability is 
increased because the yoga postures in the manualized yoga intervention are used in Hatha Yoga 
schools and can be provided by certified yoga instructors with diverse training. The stratification of 
randomization to include current PTSD diagnoses and assessment of PTSD symptoms and alcohol 
consumption prior to and during the study will provide preliminary data for use of this intervention in 
PTSD and AUD that have low parasympathetic and GABA activity. This line of research will lay an 
important foundation for hypothesis testing of other aspects of the Vagal-GABA theory, such as 
abnormalities in immune and inflammatory systems. 
 
3.0 
RESEARCH DESIGN AND METHODS 
 
3.1 
Drug/Device Information: NOT APPLICABLE 
3.2 
Type of Study: (Table 3: Individual Timeline): This is a longitudinal, efficacy randomized 
controlled trial (RCT). Veterans with MDD will be evaluated for eligibility and randomized to a 12-week 
intervention of a twice-weekly yoga and coherent breathing or walking group. As there is evidence that 
exercise improves mood in unipolar depression, the walking group will serve as an active control 
condition.(47) The policy for Common Data Elements will be incorporated into the data collection matrix. 
The Consolidated Standard of Reporting Trials (CONSORT) will be used to ensure the quality of reporting 
for this RCT.(48) The primary dependent variable will be changes in the Hamilton Depression Rating 
Scale (covering a 2 week period), and mood and anxiety scales (designed for repeated use, such that 
changes from pre to post intervention can be gauged), measured using valid and reliable psychological 
instruments obtained at screening, intervention weeks 4, 8, and 12, and before each MRS scan. The 
secondary dependent variable will be brain gamma amino-butyric acid (GABA) levels measured by 
magnetic resonance spectroscopy (MRS) in the left thalamus, the same region used in our prior studies.(9, 
53, 65) The thalamus has the technical advantage of providing high quality spectra, has a high 
concentration of GABAergic neurons, and thus a high GABA signal, in which changes can be detected. The 
thalamus is also anatomically connected to other regions of interest. GABA levels will be measured by MRS 
in 3 separate scan acquisitions: before randomization (Scan 1), after 12-weeks of the yoga or walking 
intervention (Scan 2), followed immediately by the group assigned intervention and the final scan (Scan 3). 
The third dependent variable will be respiratory sinus arrhythmia (RSA) a component of heart rate 
variability (HRV) that is an accurate index of vagal influences on the heart.RSA will be measured before 
each scan and before and after an intervention at week 4, week 8 and week 12. Medications also are 
evaluated at weeks 4, 8, and 12. Two and six month follow-up phone calls will assess depression, quantity 
of yoga practice, and barriers to practice. 

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3.3.1. Sub-Study Participation: This is not a multisite study. There will be a contract with the McLean 
Hospital for goods and services regarding the imaging component of this study. 
 
3.3.2. Study Related Procedures 
 
3.3.2.1. Instruments: The following reliable and valid instruments will be used during the screening 
interview to determine eligibility, and to monitor subjects during the interventions: 
(1) Structured Clinical Interview for DSM-5 (SCID-5) is a widely used valid psychiatric diagnostic 
instrument used to screen for Axis I disorders(66); 
(2) Columbia Suicide Safety Rating Scale (C-SSRS) is a clinically administered instrument to assess 
suicide risk(67); 
(3) Physical Activity Recall (PAR) is a widely used structured interview that yields a weekly record of 
physical activity using Metabolic Equivalents (METs), a method of assessing an individual’s level of 
physical activity in a common unit that can be calculated from a variety of activities.(55) The PAR will 
be obtained each week of the intervention in the event physical activity becomes a confounding factor. 
(4) Hamilton Rating Scale for Depression (HDRS) is a 17-item clinician administered scale 
frequently used to evaluate the effectiveness of antidepressants being assessed for FDA approval(68); 
(5) Beck Depression Inventory (BDI-II) is a 21-item self-administered instrument designed for the 
assessment of depressive symptoms, with higher scores reflecting greater severity(69); 
(6) Spielberger State-Trait Anxiety Inventory 
(STAI) is a self-administered instrument designed for 
assessment of trait anxiety (STAI-Trait) and serial 
assessments of state anxiety (STAI-State) with 
higher scores indicating higher anxiety(56); 
(7) Clinician Administered Posttraumatic Stress 
Scale (CAPS) is the gold standard clinician 
administered assessment for PTSD(70); 
(8) PTSD Check List – Civilian (PCL-C) is a 17-item 
self-administered scale corresponding to Diagnostic 
and Statistical Manual of Mental Disorders (DSM) IV 
criteria for PTSD. Because not all Veterans have 
combat experience and not all trauma is from a 
military experience, the civilian version of the PCL will 
be used. The use of the CAPS and PCL-C will allow 
assessment of severity of PTSD symptoms, and if 
present, will be modeled in statistical analyses; 
(9) Exercise-Induced Feeling Inventory (EIFI) is a 
self-administered instrument designed to assess four 
distinct feeling states associated with bouts of 
physical activity: Revitalization, Tranquility, Positive 
Engagement, and Physical Exhaustion.(57) 
(10) Pittsburgh Sleep Quality Index (PSQI) is a self-administered instrument that assesses 9 factors 
of sleep over a month period, providing a composite score, with higher scores indicating poorer sleep 
quality.(71) 
(11) Timeline Follow Back (TLFB) will be used to assess alcohol consumption for three months 
prior to screening.(72) A format similar to the TLFB will be used for nicotine consumption. 
(12) Brief Pain Inventory, an eleven point Likert Scale of pain intensity will be used to assess pain 
over the last 24 hours. 
(13) Stroop Color-Word Test is a selective attention and inhibitory cognitive control task.(74) 

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IRB Protocol Template (March 4, 2014) 
 
(14) Digit Symbol Substitution Test (DSST) is one of the frequently used tests to measure 
psychomotor slowing, and is clinically sensitive because it makes demands on several processes, 
including perception, working memory, sustained attention and visuomotor co-ordination 
(Wechsler 1997). 
(15) Child Trauma Questionnaire (CTQ) is a self-report measure of childhood trauma. (73) This 
scale will be given during the Week 4 evaluation at the end of the session. 
 
Unless otherwise stated, instruments 4-12 are used at screening, week 4, 8, and 12. In addition, the 
EIFI and STAI-State are administered before and after interventions at weeks 4, 8, and 12 and 
before each scan. The CAPS is administered at screening and at week 12. The following instruments 
are given at Imaging Session 1 & 2: 2, 5, 13, and 14. 
 
Recruitment Plan and Requirements (Table 4): Veterans will be recruited from the Bedford VA Medical 
Center (VABHCS) through posters, the internet, providing flyers to clinics, in meetings with staff, and 
recruitment at clinics affiliated with the VABHCS. Veterans who had a diagnosis of depression in the 
previous two years at the VABHCS will be identified via electronic health records. Targeted letters signed 
by the PI describing the study and inviting the Veterans to call or mail for more information will be mailed to 
Veterans. The mailing will include opt-in or opt-out card with postage and/or an opt-in or opt-out phone 
number. If a card or phone is not received within 10 days of the mailing, the identified Veterans will be 
called concerning their interest in the study. Data Access Request Tracker (DART) and VA Informatics and 
Computing Infrastructure (VINCI) will be used to identify potential subjects. 
 
3.3.2.2. If interested they will be provided information about the study and will be allowed to proceed to 
the telephone interview. These techniques have been used successfully in previous VABHCSstudies of 
yoga interventions.(59) The VABHCS Institutional Review Board (IRB) will pre- approve all forms of 
advertisement. Current VA policy does not allow email communications as part of the recruitment 
procedures. Sample size calculations, based on power analyses, require the following number of 
completers equally divided between the yoga and walking groups to test the hypotheses for each 
dependent variable: n=70 completers to test Aim 1: Depressive Symptoms and Mood Scale; n=56to 
test Aim 2: GABA changes; and n=68 to test Aim 3: RSA changes [D.20]. To meet the criteria for each 
Aim/Dependent Variable n=70 subjects will complete the intervention with n=56 having usable MRS 
data. Using data from Study 4, with consideration of changes in inclusion and exclusion criteria and 
research setting, the following recruitment schedule will be required during a 5-year study period, which 
will include over 4 years of recruitment and treatment. The required total recruitment and annual 
requirements at each stage are as follows: telephone screen=420 total or 105/year; screening 
interview=173 total or 43/year; randomization=100 total or 25/year; and completers=68 total or 18/year. 
 
3.3.2.3. Participation in Non-Assigned Group: A Retention Plan: Prior to randomization, subjects 
will be asked their group of choice (i.e., yoga or walking), as not being assigned to a group of choice 
could influence dropout rate. In order to retain subjects not assigned to their choice, subjects will be 
allowed to participate in the group they are not assigned to for 16 sessions or 8 weeks (i.e., the second 
intervention), whichever comes first, if they complete their randomized intervention and 14 week follow 
up phone call. Retention in the randomized intervention is given priority over contamination of the 6- 
month follow up with the 2nd intervention. There will be no difference in how subjects in the randomized 
intervention or second intervention will be monitored during the first eight weeks of the intervention. 
Subjects will not be paid for class attendance or evaluations during the 2nd intervention nor will they 
undergo MRS scanning. These data are exploratory. 
 
3.3.2.4. Screening: Subjects will be screened by telephone to prevent subjects from coming to the 
screening interview, only to be excluded based on information that could be obtained over the phone. 
Telephone questions do not include questions about self-harm or risk of danger. Telephone questions 
do include questions about metal and implanted medical devices that could preclude MRI scanning. If 
subjects meet telephone-screening criteria, they will be invited for an in-person screening interview at 

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the VABHCS. Subjects will be provided with the written consent to review prior to the screening 
interview. Written informed consent approved by the ENRM VA IRB will be obtained by research staff, 
defined as being able to assess participants using the Hamilton Depression Rating Scale(HDRS), the 
Clinician Administered Posttraumatic Stress Scale (CAPS) and assessing subjects for safety issues 
using the Columbia Suicide Severity Rating Scale (C-SSRS). All subjects will be consented by the PI 
who will conduct a medical history, physical exam and MRI safety screening prior to scanning. After the 
informed consent is signed, but before data collection, subjects will take a quiz to assess their 
knowledge of the study and undergo a breathalyzer test to determine Breath Alcohol Concentrations 
(BrAC). If BrAC is not zero or the subject fails to demonstrate knowledge of the study onthe quiz, 
subjects will not be allowed to participate in study activities. If the subject answers a quiz question 
incorrectly, that question will be discussed with the subject, after which the subject will initial the 
correction. If the study clinician determines the subject does not understand the protocol, including risks 
of non-conventional therapy, this would constitute reason for exclusion. The PI will review the 
inclusion/exclusion criteria to determine eligibility prior to the subject being invited to participate in the 
study. 
Potential participants are excluded for a history of psychosis, bipolar illness, and suicidal 
ideation with intent or a suicide attempt within the last year. Axis-I disorders that would interfere with 
study participation are exclusionary such as Obsessive Compulsive Disorder or Agoraphobia that would 
prevent attendance at the intervention. Alcohol Use Disorders with more than 3 DSM-IV criteria. 
Substance Use Disorders in Remission are allowed. Because inversion can increase intraocular 
pressure, individuals with a retinal hemorrhage or glaucoma cannot participate unless they receive 
clearance from their ophthalmologist. Neurologic conditions, which would effect scan results, are 
exclusionary such as stroke, seizures, brain surgery, HIV and other CNS disorders. The ability to 
participate in both interventions is required, such that a person who walks with a cane would not be 
enrolled as it would be a concern that they would either not be able to walk at 2.5 miles an hour for one 
hour or that if they did it could potentially harm them. Individuals with a history of spine surgery could be 
excluded depending on the surgery. Individual with cardiovascular or respiratory disease would require 
careful evaluation that could include a discussion with their physician. Traumatic Brain Injury with 
greater than 30 minutes loss of consciousness would be exclusionary. During the screening process, 
the potential participants have to complete a 3-minute step test (See Step Protocol). If the participant is 
unable to perform the step test, they would be excluded from the study. If during the physical exam, the 
PI is concerned about the potential participants ability to participate, they maybe asked to attempt to 
touch their toes, and to get down and then up from a yoga map or to walk the circuit that they would 
walk during the walking intervention. 
 
3.3.2.5. Inclusion Criteria: 
(1) male and female Veterans ages 18 to 65; 
(2) Fluent in English; 
(3) Understands the risks and benefits of the study as listed in the Post Consent Quiz 
(4) Females must agree to use an acceptable form of birth control [see 4.6.1.15 for acceptable 
contraception] and do not intend tobecome pregnant during the study 
(5) Females have a negative pregnancy test or a serum progesterone in the non luteal stage defined by 
less than or equal to 1.3 ng/ml prior to Scan 1. 
(6) Meets criteria for MDD on the SCID-5 
(7) Hamilton Rating Scale for Depression (HDRS) scores ≥ to 14 at screening 
(8) If subjects have been taking antidepressants that target a monoamine system, the dose has been 
stable for at least one month with no anticipated changes during the study; or no antidepressants for 
one month 
(9) If subjects have been in a stable form of psychotherapy for three months, with no anticipated 
changes in their psychotherapy during the study (this would exclude time-limited manual-driven 
therapies such Cognitive Behavioral Therapy); or no psychotherapy for three months 
(10) Reliable contact information provided 
(11) Subject weighs up to 300 lbs. at the discretion of the PI. 

IRB Protocol Template (March 4, 2014) 
 
(12) Subject has completed all required screening instruments and evaluations 
3.3.2.6. Exclusion Criteria: 
(13) History of psychosis (substance-induced psychotic disorder and psychotic disorder due to another 
medical condition are allowable if fully resolved) 
(14) History of bipolar illness 
(15) History of suicidal ideation with intent in the last year according to the Columbia Suicide Safety 
Rating Scale (C-SSRS) 
(16) Subject has a history of suicidal attempt with intent to injure in the last year according to the C- 
SSRS. 
(17) Desire to be treated for MDD with a new treatment during the study such as pharmacotherapy 
somatic therapy or psychotherapy. 
(18) A contraindication to magnetic resonance evaluation (e.g., pregnancy, a cardiac pacemaker, 
ferrous implant including shrapnel, or intrauterine devices (IUDs) with copper, claustrophobia that would 
prevent scanning, some tattoos with black ink on the head including permanent eyeliner). 
(19) Current mind-body practice (e.g., yoga, Tai Chi, Qigong, breathing practices, or meditation) 
defined as more than 6 one-hour sessions in the last 6 months. 
(20) The use of intra-uterine devices with progesterone that interferes with the ability to determine the 
non-luteal phase required to schedule Imaging Session 1. 
(21) Participates in physical exercise > 5 hours/week that is equivalent to or greater than 6 metabolic 
equivalents (METs) in intensity. 
(22) Has been treated psychotropic medications such as mood stabilizers (e.g., Valproic Acid, 
Carbamazepine, or Lithium) in the last three months. 
(23) Subject has been treated a) with a scheduled dose of anti-anxiety agents (e.g., benzodiazepines) 
or sleeping aids (e.g. Ambien or sedative hypnotics) or b) with pain medication other than Non- 
Steroidal Anti-Inflammatory Drugs (NSAIDS) in the last one month except for procedure related pain 
management (e.g., dental procedures). 
(24) Has greater than three current criteria for Alcohol or Substance Use Disorder using DSM-V criteria. 
(25) Does not have a period of 48 hours of no alcohol use on TLFB to participate in scanning 
given the effects of alcohol consumption on the GABA. 
(26) Has a neurologic condition, such as a Traumatic Brain Injury (TBI) with a loss of consciousness of 
greater than 30 min, that in the opinion of the PI could compromise subject safety or the integrity of the 
study. 
(27) Has a medical condition that in the opinion of the PI could compromise subject safety or the 
integrity of the study. 
(28) Subject would not be expected to complete the study including scheduling issues. 
(29) Has an Axis-I diagnosis, other than depression except as listed, that in the opinion of the PI would 
interfere with the subject’s participation in this study. 
(31) Has participated in another research study in the last 3 months that could affect the dependent 
variables. 
(32) Must have a period of 24 hours of no nicotine use on TLFB to participate in scanning or nicotine use 
as allowed by CSD&R Project Manager. 
(33) Subject does not agree to study procedures designed to protect against COVID-19 and the 
requirements of the virtual interventions. 
 
3.3.2.7. Randomization: Group assignments will be conducted using permuted block randomization 
with varying block sizes. Block sizes will be multiples of the number of treatments and take the 
allocation ratio into account. For this study with a 1:1 randomization of 2 groups (yoga and walking), 
block sizes will be 2, 4, and 6. Randomization will be computer generated with each randomization 
assignment put on a card in numerically labeled envelopes that will be sealed. The envelopes will be 
opened in sequential order at the time of randomization by a member of the staff that is not blinded to 
group assignment. Subjects who want more time to think about participation can be randomized within 
12 days of the last screening assessment, with the interventions starting within 7 days. 
 
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IRB Protocol Template (March 4, 2014) 
 
3.3.2.8. Group Assignment, & Blinding: All investigators are blind to group assignment prior to 
randomization. Blinding will be guided by the Cochrane Collaboration Tool for assessing risk of bias in 
randomized trials.(14) The study physicist, Dr Zuo, and Dr.Heilman, the RSA expert, will be blind to 
group assignment. Investigators who administer subjective instruments (i.e., Hamilton Rating Scale 
for Depression and CAPS) will be blind to group assignment. Subjects will fill out self-reporting mood 
and psychological scales in paper form that will be entered into the VA network by research staff. All 
VA paper documents with sensitive information will be secured in a locked filing cabinet in a designed 
VA Office. ECG data used for RSA analysis and MRS data will be labeled with a blinding code 
provided by the Data Coordinating Center, such that ECG data and MRS data are stripped of 
identifying information, including removal of study ID, date of acquisition and timing of the assessment 
(baseline, pre or post the intervention at weeks 4, 8, or 12) before being sent Dr. Heilman for analysis. 
As subjects are not blind to the intervention, there is no procedure to break the blind for clinical 
reasons. 
 
3.3.2.9. Gender Differences: While we will equally recruit both genders, due to the demographics of 
the VABHCS population, it is anticipated that more males will be recruited, which will providemuch 
needed information on the efficacy of this intervention in men. Due to the influence of menstrualcycle 
phase and associated changes of progesterone on brain GABA/Cr levels, serum levels of 
progesterone will be drawn before each scan, with progesterone levels less than or equal to 1.3 ng/ml 
being considered the non-luteal stage, a stage when GABA/Cr is not influenced by menstrual 
cycle.(75) Serum progesterone levels will be drawn at the VABHCS. 
 
3.3.2.10. Subject Compliance and Payment: Compliance with group session attendance and 
homework is important, both as an assessment of treatment acceptance and as a measure of the 
"dose" of the intervention required for symptom reduction. Attendance at the interventions will be 
recorded on an attendance sheet; home practice of yoga and walking will be recorded on the weekly 
PAR. Subjects will be paid for their time: $10 for each group session, $20 for the week 4 evaluations, 
$40 for the week 8 evaluations, $60 for the week 12 evaluations, and $40 for each MRS scan for a 
maximum of $480. 
 
3.3.2.11. Yoga Intervention: The Iyengar method uses props such that the postures can be modified 
to meet the physical abilities of the subjects. The yoga manual developed in Study 2 and 4 emphasizes 
backbends and chest opening postures consistent with the recommendations of Iyengar method for 
depression.(76) All yoga classes in the proposed intervention will be conducted according to the 
manual. Each class follows the progression of seated/reclining poses, Sun Salutation standing poses, 
spinal twists, back bends, forward bends and inversions and a deep relaxation (savasana). Consistent 
with their training, the yoga instructors are allowed to adjust poses and sequences to meet the needs of 
the subjects. The 90-minute yoga sessions include approximately 60-minutes of yoga postures, 10- 
minutes of transition and deep relaxation and a 20-minute CD of paced coherent breathing practice at a 
rate of 5 breaths per minute. In the Iyengar method, breathing practices start with exercises that use 
equal periods of inhalation and exhalation.(77) Coherence breathing uses 4 to 6 soft breathes per 
minute with equal periods of inhalation and exhalation consistent with the Iyengar method. This 
proposed study uses coherence breathing at 5 breaths per minute, an optimal rate for most people and 
a method successfully piloted in Study 4. Subjects will be instructed to attend 2 classes per week for a 
total of 24 classes. Missed classes can be made up as long as the total dose of 24 classes is not 
exceeded. Homework sessions for the yoga group are 15-minutes of yoga postures and 15-minutes of 
coherent breathing. Subjects will be provided with a yoga mat, 2 yoga blocks, 1 yoga belt, and three 
yoga blankets and a manual of the yoga sequences, and a CD of the paced breathing for at home 
practice. Additional props maybe provided for use during the virtual intervention as determined by the 
yoga instructors. The B.K.S. Iyengar Yoga National Association of the United States (IYNAUS) 
Certification Committee certifies teachers, in the B.K.S. Iyengar method for the practice and teaching of 
yoga. Ms. Elizabeth Owens, head yoga teacher for Study 2 and 4, will recruit and manage the teaching 
schedule 
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IRB Protocol Template (March 4, 2014) 
 
for certified yoga instructors with Iyengar training who have at least 5 years teaching experience. 
Instruction in coherent breathing is necessary because the positive effects on sympatho-vagal balance 
depend not only on the respiratory rate, but also on degree of effort, depth, and muscle relaxation while 
breathing. Without proper instruction, some inhale too rapidly, overfill their lungs, exhale too forcefully, 
overuse abdominal muscles, or breathe paradoxically (contracting abdominal muscles while inhaling). 
Any of these errors could increase sympathetic activation, interfere with parasympathetic activation or 
even cause hyperventilation. (16) Instructors are needed to observe subject breathing and to monitor 
and provide corrective feedback. 
The COVID-19 pandemic has resulted in all of the yoga instructors providing yoga classes virtually. In 
order to support social distancing the yoga interventions will be provided virtually through a platform 
approved by the ISSO officer, such as VA Video Connect. The yoga instructors will teach all yoga 
interventions virtually. The subjects will be required to attend the randomization, and weeks 4, 8, and 
12 interventions at the VABHCS so that equipment can be provided or collect and pre and post 
intervention data can be collected. 
 
3.3.2.12. Walking Intervention: The walking intervention was developed and used in Study 2. The 
literature documents the benefit of physical activity in the treatment of depression, such that a walking 
group can be considered an active control.(47) For the in-person walking intervention, subjects walk 
in an indoor circuit with a defined length such that the number of circuits required for a rate of 2.5 
miles an hour rate are completed in 60- minutes. Subjects are monitored so that they complete 25% 
of the required circuits every 15-minutes. The time for the group walking intervention is 60-minutes 
per group session and the homework is for 15-minutes three times a week, beyond typical walking. 
The walking group and homework are designed to match the physical activity of the yoga group (i.e., 
time spent doing yoga postures), not the length of yoga sessions, this results in the walking 
intervention taking less time than that of the yoga group, with the extra time in the yoga group due to 
the breathing exercise that does not include group interactions or physical exertion. Study staff will be 
trained to monitor the intervention and direct subjects to ensure the correct number of laps are 
completed. 
In order for the interventions to be as balanced as possible, the subjects assigned to the interventions will 
need to come to the VABHCS the same number of times as the subject assigned to the yoga intervention. 
For the virtual walking sessions, subjects will check in with research staff at the beginning and end of each 
session by video or telephone. Virtual walking sessions will also include walking at 2.5 miles an hour on a 
flat surface. During the screening process, subjects will be evaluated for stride length,such that the number 
of steps to equal 2.5 miles will be calculated. At the randomization session, subjects will be instructed on 
how to use the pedometer. Subjects will wear a pedometer for each walking session, both in-person and at 
home, the number of steps for each session will be recorded four times during the intervention and at the 
end of the homework to document the distance walked. Subjects can provide documentation of the number 
of steps by photographing the pedomenter and sending an email or text or by reporting to the walking 
intervention monitor during the session. 
 
 
3.3.2.12.1. Yoga and Walking Fidelity Assessment and Schedules: The Yoga and Walking Fidelity 
Assessments have 10 components that will be monitored for fidelity to the protocol. Each yoga 
instructor, and each walking intervention monitor will have an assessment for each quarter of study 
participation. An evaluation that has less than 100% fidelity to these 10 components will trigger a review 
of the evaluation, and a follow up evaluation will be completed within four weeks. The fidelity 
assessment can be used for in-person and virtual interventions. Four group sessions will be offered 
each week for the yoga and the walking intervention. The yoga and walking groups will be held at the 
same time to prevent blinded staff from deducing group assignment based on the time of the 
assessments. The PI may change the intervention schedule or number of session to meet recruitment 
and retention needs. 
 
3.3.2.13. Clinically Significant Abnormalities: If a participant’s tests have clinically significant 

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IRB Protocol Template (March 4, 2014) 
 
abnormalities or unexpected findings, they will be told. Specifically, females will be told if they are 
pregnant. If the serum progesterone level is not less than or equal to 0.03 ng/ml, the participant will be 
told and possibly asked to repeat the test. If there is a clinically significant finding on the anatomic MRI, 
the participant will be informed by the study PI, an M.D., with in 7 days of receipt of the report by phone 
or in person. It is McLean’s policy to call the listed physician if there is an abnormality is found during 
the MRI reading that requires immediate attention. If the participant gives written permission a copy of 
the report will be forwarded to the identified physician. 
 
3.3.2.14. Assessment for Suicidality and Safety Plan: Participants are screened weekly for 
suicidality by use of an Adverse Events sheet that they are required to turn in at the first session of the 
week, either in-person or virtual. This form may be collected by the Project Manager, or research staff. 
Due to the addition of virtual interventions, the form can be filed out by research staff over the phone. If 
there is an indication of increased depression of suicidal ideation, the project manager will inform the 
research staff or the PI that an assessment of the participant is required. There are also in-person 
evaluations by research staff at weeks 4, 8, and 12 were participants are assessed for suicidality and 
increased depressive symptoms. This study excludes individuals with a history of suicidal ideation with 
intent or a suicide attempt in the last year. This study allows suicidality without intent, which is some 
individuals is present for years. 
Assessment of suicide risk is done using the Columbia Suicide Severity Rating Scale (CSSR-S). The 
development of suicidality with intent based on the CSSR assessment will result in study withdrawal 
such that the participant can receive additional conventional treatment. If a member of the research 
staff concludes that suicidality with intent is present they will develop a safety plan with the 
participant that can include support by friends or family, the inclusion of their medical treaters, being 
taken to the VABHCS Mental Health Clinic, where admission to an in patient unit can be arranged. If 
there is a safety concern after 3:30 when the Mental Health Clinic closes, participants will be referred 
to Urgent Care or the Lahey Clinic that provides after hours emergency services for the VABHCS. If 
the participant can be managed safely as an outpatient, arrangement will be made for follow-up 
treatment. It is noteworthy suicidality decrease in Phase 1 in civilians and no subject were with 
drawn from the study for increased symptoms. 
 
3.3.2.15. Potential Problems: Recruitment goals will be assessed every 6 months. If recruitment falls 
behind schedule, study staff will meet with the staff from the numerous clinic programs at the VABHCS 
to inform them of this research opportunity. If enrollment in the imaging component is behind due to 
nicotine use causing more than 10% of exclusions in a 6 month period, the inclusion criteria regarding 
smoking will be increased to include all subjects, regardless of abstinence. 
 
3.3.2.16. Acquisition of Mood Scales and Psychological Tests: Participants will enter self-report 
mood and psychological scales onto paper forms. Investigator completed psychological mood and 
psychological scales will be entered into the REDCap database by research staff. The computers ued 
for storage of mood scales and psychological tests will be VA computers. 
 
3.3.2.17. Acquisition and Analysis of GABA MRS and MRI images: Acquisition and Analysis of 
GABA MRS and MRI images: All MRS/MRI data will be collected on a Siemens 3T scanner at the 
McLean Imaging Center (MIC), McLean Hospital, Belmont, MA. High-resolution, 3D T1-weighted 
anatomical images will be acquired to prescribe the voxel with anatomical precision and 
reproducibility. Placement algorithms developed in our prior studies will be used for consistent voxel 
placement, based on anatomic landmarks, in the left thalamus (2x2x3 cm). A single-voxel proton 
MRS GABA difference-editing technique, MEscher-GArwood Point-Resolved Echo Spectroscopy 
Sequence – MEGAPRESS, will be used for spectral acquisition, as it permits detection of high-quality 
GABA spectral data. MEGAPRESS allows for collection of TE 68ms PRESS spectra for the detection 
of other important metabolites such as creatine, NAA, choline and glutamate. MEGAPRESS is a well- 
established GABA spectral difference-editing method that isolates the GABA doublet at 3.00 ppm in 
vivo.(78) When used in conjunction with PRESS (point-resolved spectroscopy). Resting state data 

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has been collected at pre-randomization in Study 4, and now can be collected pre and post the 
intervention to assess for changes in resting state over the course of the study. A resting-state fMRI 
scan of 8-10 minutes will complete the one-hour scanning. If time constraints require, the resting- 
state fMRI scan will not be done. Minor adjustments in the protocol within the safety parameters that 
do not change the science of the study are allowed. 
 
As discussed with VACO, if there is a problem acquiring scans at the McLean Hospital (e.g. system 
upgrade) the study can proceed without the imaging component without a protocol deviation.  
 
3.3.2.18. Acquisition and Analysis of PNS Measures: This protocol was used in Study 4. The skin 
will be cleaned with an alcohol wipe prior to placement of three electrodes at heart level in the left mid- 
axillary line, above the umbilicus, and under the right clavicle. ECG data will be collected using a GGP 
Biolog IBI monitor (UFI, Morro Bay, CA) in three consecutive phases: (1) resting, while sitting quietly for 
5-minutes; (2) step, a 3-minute challenge using the YMCA Step protocol; and (3) recovery, a 5-minute 
sitting period. The YMCA Step Test is a standardized test used for 30 years as part of the YMCA 
Fitness Testing and Assessment Manual.(81) The Official YMCA test consists of stepping up and down 
on a 12-inch step in time to a 96 per second metronome for a total of 26 step cycles per minute. As in 
Study 4, the test will be modified to use an 8-inch step to increase subject ease. The step challenge is 
designed to remove the vagal brake such that RSA deceases and then increases during the recovery 
period.(82) ECG data will be obtained prior to randomization, and pre- and post-intervention at week 4, 
week 8, and week 12, and before each MRS scan. The ECG data will be acquired using a Faros 
Bittum 180 and software approved and installed on VA computers htransferred to a VA computer using 
the 3991xGPP Biolog progam (see attached Faros Bittum manual). The data will be blinded and sent 
to Dr. Heilman for analysis. 
 
3.4. Data Collection 
 
3.4.1. Discussion of VA research activities and data for non-VA research activities and non-VA 
data: All activities conducted under this study are for research proposes. The VABHCS is the 
coordinating center through which all recruitment, advertisements, participant assessments, and 
interventions associated with this research project will occur with the following exceptions. There will be 
a contract with the McLean Hospital for the acquisition, and analysis of the MRI data. There will be an 
contract with Dr. Keri Heilman for the analysis of the ECG data. The contracts and agreements will 
clearly state that the data acquired and analyzed are good and services and belong to the VA. 

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3.4.2. Data collection, storage, access, use, disclosure, and analysis: 
 
3.4.2.1. 
Describe all data collection activities for VA research: 
The collection of imaging data at the McLean Hospital was approved by VACO. There are three 
dependent variables: 1) Hamilton Depression Rating Scales, 2) Thalamic GABA levels, and 3) 
Respiratory Sinus Arrhythmia (RSA). 
3.4.2.1. 
Psychological Scales: All data from screening and intervention assessments will take 
place at the VABHCS and will be put into REDCap. Evaluations at the McLean Hospital will be put 
into REDCap using a VA issued laptop. 
3.4.2.2. 
Spectroscopic Data: All magnetic resonance imaging (MRI) and magnetic resonance 
spectroscopy (MRS) data used for analysis of GABA levels will be collected and analyzed at the 
McLean Hospital under a contract that will state that the data collected under the contract belongs to 
the VABHCS. The analyzed data will be transferred via secure electronic means for incorporation in 
the final data set. All individuals scanned at the McLean Hospital are checked in using their name, 
address and social security number. 
3.4.2.3. 
Formal Reading of MRI Study: It is the policy of the McLean Hospital that all research 
subjects have a VA brain scanning sequence that is formally read by a person qualified to read MRI 
studies. This formal reading does have the subject name on it. The data that will be used for the study 
will only be marked with the subject ID. 
3.4.2.4. 
RSA Data: Electroencephalogram (ECG) data will be collected 9 times during the study, 
three times at the McLean Hospital prior to each scan and before and after evaluations at weeks 4, 8, 
and 12 at the ENRMH. This data will be collected using a hand held device, a Bittum Faros, and 
transferred to a VA laptop computer where it will be processed and blinded before being sent through 
secure electronic means to Dr. Keri Heilman, Co-Investigator for analysis. Dr. Heilman will then sent the 
analyzed data back for incorporation into the final database. The software that will be used to transfer 
the data from the Bittum Faros will be put on the VA computers by the VABHCS Information 
Technology (IT) staff. 
 
3.4.2.5. 
Storage: Data will be stored in the REDCap database that will be created within the VA 
system, scan data will be stored in the locked McLean hard drives which as backed up regularly, the 
original ECG recordings will be stored on the VA network in a designated file. The ECG data sent to Dr. 
Heilman will not contain HIPPA identifiers, when the data is returned it will be saved on a VA computer. 
The final data set will incorporate REDCap, imaging and RSA data. The management of the REDCap 
database and merging of the other databases into the final data will be done by the study statistician 
and Healthcare Specialist in accordance with VA regulations. The desktop computer will be stored in 
the Project Manager’s Office, The laptops will be stored in Dr. Streeter’s and the research staff’s office 
and will be taken to the McLean Hospital as needed. Dr. Streeter will also be Teleworking such that 
she will take her computer with her to her home and appointments, this computer will be encrypted. 
Papers with personal information will be kept in the offices of Dr. Streeter, the Project Manager and 
research staff in locked filing cabinets when not in use. Data regarding scan acquisition will be kept in a 
locked filing cabinet at the McLean Hospital or on secure servers. A Final Copy of the Database will be 
stored at the VABHCS on the VA network. One copy will be stored on the PI’s VA laptop, a third copy 
will be provided to the study statistician. The database is owned by the VA. 
3.4.2.6. 
Access and Use: Only member of the research team and individuals providing goods 
and services through contracts or Interagency Personnel Agreements (IPAs) will have access to the 
data. When study personnel are no longer members of the research team, they will no longer have 
access to the study data. The exception would be through the Data Management Access Plan 

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submitted with the Merit application. 
3.4.2.7. 
Statistical Analysis: The data from REDCap, MRI/MRS and RSA will be merged in to a 
final dataset for analysis by study statistician. The final dataset will be a Limited Data set in which the 
only HIPPA identifier will be the date of data collection. This data collection and analysis plan was 
included in the submitted proposal that was approved by the VACO, such that they were aware that 
data was going to be acquired and transferred outside of the VABHCS. 
3.4.2.8. 
Security Measures: There are security measures in place at the VABHCS, the Data 
Coordinating Center, and the McLean Hospital. 
3.4.3.1. 
Will VA data be combined with non-VA data: Not Applicable, all data is VA data, as 
data collected at the McLean Hospital is obtained under a contract. VACO has approved for Dr. 
Streeter to be off-site at the McLean Hospital to oversee the imaging component of the study. Dr. 
Streeter has appointments at the VABHCS, the McLean Hospital, and Boston University School of 
Medicine, the site of the Data Coordinating Center. VACO has approved Dr. Streeter to have off-site 
time, such that she can use combined data while on VA time. 
3.4.3.1. 
Identify any VA research activities occurring at non-VA sites: All imaging acquisition 
and analysis will take place at the McLean Hospital. Dr. Heilman will conduct analysis of the ECG s for 
RSA off site with data that does not contain personal identifiers but will contain the data of acquisition. 
The study statistician will merge the data from REDCap, analyzed spectroscopic data, and RSA data 
into a final dataset. 
3.4.3.2. 
Existing Protocols: Not Applicable 
3.4.3.3. 
Provide a copy of any memorandum of understanding (MOU) with the non-VA 
entity describing data ownership or data security arrangements for the “collaborative” study: 
The McLean Contract provided for IRB approval and contains information regarding data ownership 
and data security. The contract for data management and data security that will contain Memorandum 
of Understanding (MOU) or contract describing the data ownership and security arrangements will be 
submitted to the IRB for approval before the data management and analysis can proceed. 
3.4.3.4. 
This protocol does not collect non-VA research. 
3.4.3.5. 
Uses and Disclosures of Protected Health Information (PHI): The data will only be 
used for research. The REDCap, ECG and MRI databases will only identify participants by their subject 
ID. Of the 18 HIPPA identifiers, only the date of date collection will be included in the Limited Final 
Database. The Final Database will include the dates of the assessment, which would not pose a 
reasonable risk of allowing the subject to be identified. There will be a separate database in REDCap 
with contact information, such that the PI or other research staff can contact subjects. The protections 
relating to HIPPA will be followed. 
3.4.3.6. 
Researchers and Study Team Members: All research team members will be identified 
in the IRB approved study. Study team members are defined as non-VA employees who are involved 
in the contracts. Only researchers and study team members will have access to the data with the 
exception of the required regulators. While at the McLean Hospital, member of the McLean staff, who 
are not part of the research team, will interact with subjects at check in. 
3.4.3.7. 
The Principal Investigator, Dr. Chris Streeter will have at the start of the study the 
following appointments: 1) a 3/5th appointment at the VABHCS VA; 2) an appointment at the  
Boston University School of Medicine for which she is compensated for work that is non-overlapping 
with this study; and 3) a without compensation appointment at the McLean Hospital. 
(i) 
VA duties: The PI’s 3/5th VA appointment includes clinical time as a member of the 
department of psychiatry at VABHCS and her duties as the PI of this study 
(ii) 
VA duty locations: The VA duty locations are the VABHCS for for clinical duties related 
to VABHCS and the conduct of this study. 
(iii) 
VA tours of duty or time allocations: The PI’ 3/5th VA appointment is equivalent to 24 hours a 
week plus a 30 minute meal break for period of work over 4 hours. This study will be run using the 
same structure that has worked for the civilian version of this study. In order to provide intervention 
times such that individual who are and are not employed can participate in the study, the interventions 
will be conducted from 8 am to 9:30 am and from 5 pm to 6:30 pm on Monday and Wednesday, the 
days of the intervention can be changed or additional sessions added as needed. The week 4, 8 and 12 

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evaluations that take place before and after the interventions and take around 45 minutes. To 
accommodate this schedule, Dr. Streeter’s VA tour of duty will be from 7:30 am to 8:00 pm on Monday 
and Wednesday which includes a 30 minute meal break. After subtracting 30 minutes for lunch each 
day this results in two 12-hour tours, 24 hours or 3/5th time. 
(iv) 
Issues related to data ownership: All data collected at the McLean Hospital will be collected 
under a contract which will state that the data belong to the VABHCS. All data analyzed by Dr. 
Heilman belongs to the VABHCS. Research information protection and data security requirements. 
The protocol approved by Veterans Administration Central Office (VACO) included permission for Dr. 
Streeter to be off site to oversee the collection and analysis of data at the McLean Hospital and for Dr. 
Heilman, Dr. Zuo, the study physicist to have access to a Limited Data Set. HIPPA identifiers will 
remain within the REDCap database, the VA Medical Health Record and the McLean Medical Health 
Record. 
 
3.5. Analysis Plan 
3.5.1. Data Management and Analysis Plan: The study statistician and Healthcare Specialist will be 
responsible under a contract for data management and statistical analysis for the project.The Healthcare 
team will convert the REDCap shell from an earlier study and provide the monitoring reports, create of 
the final database. The study statistician will oversee the statistical analysis with the help of the 
Healthcare Specialist. Descriptive statistics will be generated for baseline data and distributions 
between the yoga intervention and walking intervention will be compared using two-sample t-tests and 
chi-square or Fisher’s exact tests toassess effectiveness of randomization. Descriptive statistics 
include means and standard deviations forcontinuous variables and group sizes and proportions for 
categorical variables. All analyses with be performed at the α = 0.05 level of significance. Should 
groups have unequal distributions at baseline, relevant variables will be incorporated in subsequent 
linear models that will be used to examine intervention efficacy. In order to address the study aims of 
assessing the relative longitudinal change independent variables between yoga intervention and 
walking intervention groups over time, data will be collected at four time points, pre-randomization, and 
at weeks 4, 8, and 12. Mixed linear model analyses will be preformed focusing on the group x time 
interaction for the primary effects to be statistically modeled. These analyses will allow for inclusion of 
data at differing numbers of time points per subject if needed. We will examine different within-subject 
correlation structures for our interaction model and determine the optimal structure by comparing 
Akaike information criteria (AIC) values. The correlation structure that yields the lowest AIC will be 
adopted. Subjects who change antidepressant medications during the study will be included in intent to 
treat analysis, but will be co-varied for medication changes. Potential cohorts effects will be statistically 
addressed by controlling for group profiles, including group size, breakdown by sex of attendees, 
instructor, group parameters such as individual or group sessions, and group cohesion/overlap. 
 
3.5.1.Data Managment: The study statistician will provide leadership and expertise in data collection, 
management and analyses, encompassing database design, implementation of quality control 
procedures, technical support, and data sharing. A close interaction between data management staff 
and research team members will result in procedural consistency, efficiency of the study-wide 
systems, and ultimately produce the highest quality data. Data collection strategies will include web- 
based assessments using VA REDCap software. When possible, data will be collected on desktop 
computers, or laptops, negating the need for double entry. Electronic forms will include required fields, 
skip patterns, and validation resulting in more complete and “cleaner” data at capture. All participants 
will be assigned a unique identifier (ID) and personal identifiers that must be collected and used for 
consent and follow up will be stored in a separate database apart from study data. Analysed data from 
McLean Hospital and Dr. Heilman will be provided via a VA approved system (e.g., AZURE) 
 
3.5.1. Analysis of Hypotheses (Aim 1: Hypotheses I, Aim 2: Hypotheses 2, Aim 3:Hypothesis 3) 
Hypothesis I. a. Depressive Symptoms: The yoga group will have decreased depressive 
symptoms compared to the walking group. There will be a significant within-group change (decrease) in 

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depressive symptoms over time, using a mixed linear model analogue of repeated measures analysis- 
of-variance with independent groups in MDD subjects assigned to the yoga group. 
Hypothesis 1. b. Mood Scales: There will be a greater improvement in mood (as measured by the 
EIFI and STAI), and sleep (as measured by the PSQI) in the yoga group compared to the walking 
group. 
Hypothesis 2. a. GABA: GABA levels will increase in the yoga group. There will be a within-group 
change in GABA levels over time using a mixed linear model analogue of repeated measures analysis- 
of-variance with independent groups in MDD subjects assigned to the yoga group. 
Hypothesis 2. b. Group Differences: There will be a between-group difference in GABA levels 
over time using linear modeling comparing the yoga group with the walking group. 
Hypothesis 3. a. RSA: There will be tonic increase in post intervention recovery RSA in the yoga 
but not the walking group. 
Hypothesis 3. b.: There will be an increase (from baseline to end of week 12) in the difference 
between pre- and post-intervention in the rest and the recovery RSA in the yoga group. 
Hypothesis 4. a. Exploratory Concurrent Antidepressants: The yoga group but not the walking 
group will have a decrease in depressive symptoms regardless of antidepressant treatment at the 
beginning of the study. 
Hypothesis 4. b. Exploratory PTSD Symptoms: If PTSD symptoms are present at screening, 
there will be a greater decrease in PTSD symptoms in the yoga compared to the walking group as 
measured by CAPS and PCL-C. 
Hypothesis 4. c. Resting State Connectivity will increase over the course of the study with greater 
increases in the yoga group. 
Hypothesis 4. d. Executive Function will increase over the study as measured by the Stroop, Digit 
Symbol test and fMRI activation. 
 
3.5.2. Power Analysis for Depressive symptoms and Mood Scales: Based on the power analyses 
below, in order to achieve adequate power for all analyses, n=70 subjects, n=35 in the yoga and n=35 
in the walking intervention must complete the interventions. Using Study 4 data in subjects with MDD in 
the LDG, the repeated measures ANOVA for EIFI scores from screening to the completion of the 
intervention did not reach statistical significance in the group of n=15 (baseline: 3.1 ± 2.2 and week-12: 
3.9 ± 2.7, F1,13 = 1.097, p = 0.31), a post hoc power analysis was conducted, which determined that the 
achieved effect size was f =.281, with 52% power. Based on this same effect size, an a priori power 
calculation was conducted, which determined that n=35 subjects would be needed to detect significant 
differences for BDI-II, STAI, and EIFI scores from screening to the completion of the intervention, with 
effect size f =.281, with < =.02 to account for multiple comparisons, and 81% power. As the power 
analysis for the EIFI requires the largest number of subjects (n=70), which will be used to determine 
sample size. In order for 70 subjects to complete the intervention, assuming a 20% drop out, 84 
subejcts will be randomized. 
 
3.5.3. Power Analysis for GABA: Consistent with our analytic plan for the longitudinal questions at- 
hand, we can compute the statistical power for Hypothesis II an analysis sample of n=26 subjects in 
each of the study groups using the mixed linear model analogue of repeated measures analysis-of- 

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variance with independent groups. We used nQuery Advisor, version 7.0 for these computations. Using 
GABA level as the dependent variable of interest and employing the observation from Study 2 in which 
the walking intervention showed no change in GABA over the 12-week intervention, it is predicted that 
GABA levels will not change over time in the walking group. We hypothesize that the yoga group will 
show mean GABA/Cr = 0.28, 0.285, and 0.32 at the three study time points and that the walking group 
will show mean GABA/Cr = 0.28, 0.28, and 0.28. Assuming a common standard deviation at each time 
point of 0.06 and a within-subject correlation of 0.65, we will have 80% power to detect the above noted 
pattern of repeated mean GABA/Cr with a two-sided alpha of 0.05. With the same data configuration, 
we will have 85% power should the within-subject correlation be as high as 0.70. 
 
3.5.4. Tonic changes in Recovery RSA from screening to 12 weeks: The GEE of changes in 
recovery RSA in the full cohort (n=27) over 12 weeks was significant in Study 4; however, only the 
recovery RSA values in the smaller LDG (n=14) were used for the power analysis, as this reflects the 
dosage that will be utilized in the proposed study. While the repeated measures ANOVA did not reach 
statistical significance in the LDG (screening mean = 7.7 ± 2.5 and 12 week mean = 8.2 ± 2.9, F1,12 = 
1.094, p = 0.315), a post hoc power analysis was conducted, which determined that the achieved effect 
size was f = 0.291, with 52% power. Based on this same small to medium effect size, an a priori power 
calculation that includes two groups (yoga and walking) was conducted, which determined that n=34 
subjects would be necessary to detect significant differences in RSA at effect size f = 0.291, with 90% 
power. 
 
3.5.5. Acute changes in RSA at 12 weeks (pre and post yoga intervention): Half way through Study 
4, an RSA collection was added prior to the 12-week yoga intervention, which allowed acute changes in 
RSA to be calculated. An ANOVA of rest stage changes reached statistical significance; 12-week pre 
intervention resting mean = 4.9 ± 18 and 12-week post intervention resting mean = 5.7 ± 1.6 (F1,14 = 
11.056, p = .005). A post hoc power analysis was conducted, which determined that the achieved effect 
size was f =.858, with 99% power. Based on this same large effect size, an a priori power calculation 
was conducted, which determined that n=10 subjects would detect significant differences at effect size f 
=.858, with alpha = .01 and two groups (yoga and walking) would achieve 90% power. An ANOVA of 
rest stage changes reached statistical significance; 12 week pre intervention recovery mean = 6.7 ± 2.4 
and 12 week post intervention recovery mean = 8.1 ± 3.4 (F = 3.819, p= 0.070). A post hoc power 
analysis was conducted, which determined that the achieved effect size was f = 0.504, with 96% power. 
Based on this same large effect size, an a priori power calculation was conducted, which determined 
that n=20 subjects would detect significant differences at effect size f = 0.504, with α = .01 and two 
groups (yoga and walking) would achieve 90% power. 
 
4.0. HUMAN SUBJECTS 
 
4.1. General Characteristics: Subjects will be 18 to 65 year old Veterans with a diagnosis of Major 
Depressive Disorder. Anxiety Disorders, including Post Traumatic Stress Disorder, and Alcohol or 
Substance Abuse Disorder with three or less criteria met according to Diagnostic and Statistical Manual 
of Mental Disorders 5 (DSM-5) criteria will be allowed as long as these disorders will not interfere with 
the subject’s safe participation in the study or interfere with the integrity of the study. While there are 
more males than females being served by the hospital, more women than men chose to do yoga, such 
that the percentage of female participants maybe greater than the percentage of females served by the 
hospital. 
 
4.2. Inclusion of Vulnerable Subjects and Special Populations: Children are included as 18 to 21 
years olds. Enlistment in the Armed Services requires an individual is at least 17 years old with 
parental consent. As enrollment, discharge and registration at the VABHCS for care would take at 
least a year, such that this study of Veterans cannot include subjects under 18 years old. This study 
will recruit and include males, females and minorities. The Targeted Enrollment Table is based on the 
following estimates: Females 10%, Hispanics 6% and African Americans 11%. This study includes 

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individuals with mental illness; however, the inclusion/exclusion criteria are such that these individual 
would have the capacity to understand the risks and benefits of the study such that they could make an 
informed decision. This study does not enroll pregnant females as confirmed by a pregnancy test and 
the requirement to use an acceptable form of birth control. Due to the possibility of therapeutic 
misperception by the participants as to what is research and what is clinical care, it is clearly stated 
that this is a research study that does not overlap with clinical care and the participants can withdraw at 
any time with no effect on their clinical care. 
 
4.3. Inclusion of Incompetent Subjects: Individuals without capacity will not be enrolled in this study. 
This study does enrolling individuals with psychiatric illness who will based on the inclusion and 
exclusion criteria have the capacity to consent. 
 
4.4 Inclusion/Exclusion Criteria 
 
4.4.1. Inclusion Criteria: 
1 male and female Veterans ages 18 to 65; 
2 Fluent in English; 
3 Understands the risks and benefits of the study as listed in the Post Consent Quiz 
4 Females must agree to use an acceptable form of birth control [see Human Subjects] and do not 
intend tobecome pregnant during the study 
5 Females have a negative pregnancy test or a serum progesterone in the non luteal stage defined by 
less than or equal to 1.3 ng/ml prior to Scan 1. 
6 Meets criteria for MDD on the SCID-5 
7 Hamilton Rating Scale for Depression (HDRS) scores ≥ to 14 at screening 
8 If subjects have been taking antidepressants that target a monoamine system, the dose has been 
stable for at least one month with no anticipated changes during the study; or no antidepressants for 
one month 
9 If subjects have been in a stable form of psychotherapy for one month, with no anticipated changes in 
their psychotherapy during the study (this would exclude time-limited manual-driven therapies such 
Cognitive Behavioral Therapy); or no psychotherapy for 1 month 
10 Reliable contact information provided 
11 Subject weighs up to 300 lbs. at the discretion of the PI. 
12 Subject has completed all required screening instruments and evaluations 
 
Exclusion Criteria: 
13 History of psychosis (substance-induced psychotic disorder and psychotic disorder due to another 
medical condition are allowable if fully resolved) 
14 History of bipolar illness 
15 History of suicidal ideation with intent in the last year according to the Columbia Suicide Safety Rating 
Scale (C-SSRS) 
16 Subject has a history of suicidal attempt with intent to injure in the last year according to the C- SSRS. 
17 Desire to be treated for MDD with a new treatment during the study such as pharmacotherapy somatic 
therapy or psychotherapy. 
18 A contraindication to magnetic resonance evaluation (e.g., pregnancy, a cardiac pacemaker, ferrous 
implant including shrapnel, or intrauterine devices (IUDs) with copper, claustrophobia that would 
prevent scanning, some tattoos with black ink on the head including permanent eyeliner). 
19 Current mind-body practice (e.g., yoga, Tai Chi, Qigong, breathing practices, or meditation) defined as 
more than 3 one-hour sessions in the last 3 months. 
20 The use of intra-uterine devices with progesterone that interferes with the ability to determine the non- 
luteal phase required to schedule Imaging Session 1 
21 Participates in physical exercise > 5 hours/week that is equivalent to or greater than 6 metabolic 
equivalents (METs) in intensity 
22 Has been treated psychotropic medications such as mood stabilizers (e.g., Valproic Acid, 

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Carbamazepine, or Lithium) in the last three months. 
23 Subject has been treated a) with a scheduled dose of anti-anxiety agents (e.g., benzodiazepines) or 
sleeping aids (e.g. Ambien or sedative hypnotics) or b) with pain medication other than Non-Steroidal 
Anti-Inflammatory Drugs (NSAIDS) in the last one month except for procedure related pain 
management (e.g., dental procedures). 
24 Has greater than or equal three current criteria for Alcohol or Substance Use Disorder using DSM-V 
criteria 
25 Must have a period of 48 hours of no alcohol use on TLFB to participate in scanning given the 
effects of alcohol consumption on the GABA. 
26 Has a neurologic condition, such as a Traumatic Brain Injury (TBI) with a loss of consciousness of 
greater than 30 min, that in the opinion of the PI could compromise subject safety or the integrity of the 
study. 
27 Subject with a medical condition would not be expected to complete the study, or their participation 
would be jeopardized subject safety or the integrity of the study 
28 Subject would not be expected to complete the study including scheduling issues. 
29 Anxiety disorders with current symptoms that would impair participation in the study (e.g., Obsessive 
Compulsive Disorder (OCD) or agoraphobia that would prevent intervention attendance, Post 
Traumatic Stress Disorder with dissociation or flashback that could be triggered by the interventions 
30 Has an Axis-I diagnosis, other than depression except as listed, that in the opinion of the PI would 
interfere with the subject’s participation in this study 
31 Must have a period of 48 hours of no alcohol use on TLFB to participate in scanning given the effects of 
alcohol consumption on the GABA. 
32 Has participated in another research study in the last 3 months that could affect the dependent 
variables 
33 Subject does not agree to study procedures designed to protect against COVID-19 and the 
requirements of the virtual interventions. 
 
 
• 
Race, Gender, Ethnicity and HIV Status 
Potential participants will not be excluded based on their race, religion, or ethnicity. HIV positive status 
will results in exclusion due to the potential effect on the imaging data and thus the integrity of the 
study. Exclusion due to HIV would be made under exclusion number 20: has a neurologic or medical 
condition that in the opinion of the PI could compromise subject safety or the integrity of the study. 
There is no exclusion related to gender. However a transgender individual taking hormones would 
result in exclusion due the inability to determine the non-luteal phase of the menstrual cycle such that 
the first imaging session could be scheduled. 
 
• 
Fairness of Recruitment 
Recruitment will be done by flyers, contacting individuals who have the diagnosis of depression, 
meetings with unit teams and staff. All interested subjects will be screened and included or excluded 
using the stated criteria. 
 
4.5. Recruitment 
 
4.5.1.1. Subject Identification and Pre-Enrollment Screening: Subjects will be identified through 
flyers, advertisements, interactions with VA staff and through the electronic database. Potential 
subjects who have not called asking for information will first be contacted by mail which will include 
instructions on how subjects can opt in or opt out by calling a phone number. The telephone interview 
form shows the information that will be collected and does not include a social security number. An opt 
out approach is requested such that subjects can be contacted and given the opportunity to ask 
questions if they are undecided on whether they wish to participate. 
 

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4.5.1.2. Consent for Recruitment and/or Screening: Subjects will be read the telephone informed 
consent over the phone, or if they request a copy will be mailed to them. Subjects are provided with a 
copy of the written informed consent by mail if requested or are given the opportunity to read the written 
informed consent or prior to the screening interview. To avoid coercion, subjects are told that they can 
withdraw at any time without jeopardy to their medical care. To avoid therapeutic misconception, 
subjects are told that this is a research study that it is not part of clinical care. A request for a waiver of 
documentation of informed consent is requested for the telephone informed consent. Participants will 
sign, date and put the time of the consent on the informed consent at the beginning of the screening 
interview prior to the collection of any data, after the consent has been reviewed by research staff and 
subjects have had the opportunity to ask questions. The research staff, defined as being trained to use 
the Hamilton Depression Rating Scale (HDRS), the Clinician Administered Posttraumatic Stress Scale 
(CAPS) and assessing subjects for safety issues using the Columbia Suicide Severity Rating Scale (C- 
SSRS) will obtain the informed consent, and will also sign, date and put the time on the consent on the 
informed consent at the time of the informed consent. The study physician may obtain the initial 
informed consent, if not he/she will sign the informed consent when the subject is consented with 
regards to the medical/imaging aspect of the study. There is a clinical reporting form titled Enrollment 
Progress note that is filled out at the time of the consent. After the consent, subject take a quiz to 
determine if they understand the risks, benefits and procedures related to the study. This process is 
conducted in a private room to ensure subject privacy. As subjects have the capacity to consent, there 
will be no use of surrogates. The subject will be given a copy of the signed informed consent. 
 
4.5.2. Enrollment 
4.5.2.1. 
HIPPA Authorization: Enrolled subjects will sign a HIPAA authorization form to 
provide the following information : Information from VA Health Records such as diagnoses, 
progress notes, medications, lab or radiology findings; Specific information concerning: 
alcohol abuse, drug abuse, HIV; Demographic Information such as name, age, race; 
Questionnaire, Survey, and/or Subject Diary; Imaging Data, electrocardiogram data, physical 
exam, blood and urine tests. 
4.5.2.2. Waiver of HIPAA Authorization: Waiver of the HIPAA authorization requirement is required for 
recruitment purposes only. HIPAA authorization will be sought from participants prior to 
enrollment.  The information requested is for screening and recruitment to achieve 
enrollment. We will also conduct brief screenings with veterans determine eligibility.  The 
research uses the telephone screening information to identify subjects to be invited for an in-
person screening interview. Recruitment also requires the assistance of DART, and VINCI 
that are approved in the protocol to further identify subjects.  Data regarding recruitment will 
be obtained from DART, CARRI/JVL, CAPRI/JVL/Remote Medical Record/CPRS and VINCI, 
with national access to the medical record/EHR through CAPRI. The telephone interview 
form is designed such that all identifiable information is listed on the first page. All medical 
and study information is listed on the subsequent pages that are identified by subject ID. At 
the end of the interview, the first page is separated from the reset of the documents. All 
personal identifiable information is kept in a separate file from information identified by the 
subject ID. JVL and CPRS may be accessed by the study PI to determine eligibility. 
Recruitment: Data will be transferred from VINCI to local server by using VINCI download tool 
to the Custodians at the VA Bedford Healthcare system. All research staff have VA issued 
computers and VA emails. The data will not be taken off the VA computers except to print 
mailing labels for recruitment. Data will not be taken outside of the VA firewall. 
 
4.5.2.3. Informed Consent: There are two stages in the informed consent process. The first is 
consent for a telephone screening for which a waiver of written informed consent was granted. 
Potential subjects are read an approved consent form that contains all the components of a written 
informed consent. The member of the VA research team that obtains the telephone informed consent 
will sign and date the telephone informed consent form. After the participant agrees to participate in 

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the telephone screening they are asked the questions on the telephone screening questionnaire by 
any member of the Research Staff. If the participant pasts the telephone screening, they will be invited 
for an in-person screening interview and/or given the option of being consented with docu-sign that 
this study has been approved to use. The advantage of docu-sign allows much of the screening 
procedures to be conducted over the phone or VVC, such that social distancing between potential 
subjects and research staff is decreased. The initial written informed or docu-sign consent maybe 
obtained by a research staff, defined as an individual able to use the Hamilton Depression Rating 
Scale (HDRS), theClinician Administered Posttraumatic Stress Scale (CAPS) and assessing subjects 
for safety issues using the Columbia Suicide Severity Rating Scale (C-SSRS). Both the participant 
and the research staff will sign, date and place the time of the signature on the written informed 
consent or using docu-sign. This will allow the screening interview to proceed. Prior to scanning, a 
physician (e.g., the study PI), will complete theinformed consent process with the subject with regards 
to the medical and scanning aspects of the study at which point they will sign, date and place the time 
of the signature on the written informed consent. The written informed consent or docu-sign is 
obtained in a private office such that the participant can ask questions. In all instances, the participant 
would have to have an in-person evaluation for a physical exam and for the informed consent 
regarding imaging to be signed by the study PI prior to advancing to an imaging session. During the 
telephone screening for which a waiver of written informed consent is requestedthe subjects may give 

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consent to be contracted by AZURE so that they can be provided with a copy ofthe written informed 
consent to review before the in person interview. 
 
4.5.2.4. Definition of Enrollment and Randomization: Participants are considered enrolled in the 
study when they give oral consent to the telephone interview. There is no requirement for written 
consent to the telephone interview. Participants are considered enrolled in screening after they sign 

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the written informed consent. Participants are considered randomized when they participate in their 
first intervention. All subjects who consent to the telephone interview are given a subject ID number 
and are listed on the master database that links the subject name to their subject ID. The telephone 
interview has two parts. The first page the telephone interview contains identifying information; this 
page is separated from the interview after the subject is assigned a subject ID that is used to mark the 
telephone questionnaire. The information in the telephone informed consent does not collect 
information regarding drug abuse, alcohol abuse, HIV or sickle cell. 
 
4.5.2.5. 
Recruitment Procedures: Subjects will be screened by telephone to prevent 
subjects from coming to the screening interview, only to be excluded based on information that 
could be obtained over the phone. Subjects can be provided a copy of the telephone consent 
to read prior to the telephone screen if requested. If not they will be read the telephone 
informed consent over the phone. A waiver of written consent is requested for the telephone 
screen as it covers questions that could be asked during a routine medical appointment and 
does not include questions about self-harm or risk of danger. The guide for the telephone 
interview is provided as a Clinical Report Form (CRF), this guide consist of a page with 
identifying information such as telephone number, email and address so that information can 
be sent to the subject. On a different form identified by a subject ID is the text that guides the 
interview and the information that will be collected. The request for a waiver of written consent 
for the telephone interview was granted for Phase 1 and Phase 2 of the civilian study on which 
this study is based. In previous studies, approximately 1 out of 3 subjects advances from the 
telephone interview to the screening interview. The telephone interview prevents potential 
participants from having to come for an interview when the telephone screen is guided by a 
form that does not include social security numbers. Telephone contacts will be done on 
subjects who respond to advertisements or letters. If subjects meet telephone-screening 
criteria, they will be invited for an in-person screening interview at the VABHCS. Subjects will 
be provided with the written consent to review prior to the screening interview. Written 
informed consent approved by the VABHCS IRB will be obtained by research All subjects will 
be consented by the PI, a M.D., who will conduct a medical history, physical exam and MRI 
safety screening prior to scanning. There are two staff signature lines on the informed consent, 
as the research staff can perform the screening interview except for the procedures that must 
be performed by an M.D. All consent procedures will take place in a room that will ensure the 
participants privacy. Advertisements or letters being sent to potential participants will be 
submitted to the IRB for approval prior to use. 
 
4.5.2.6. 
Creation of a VHA Health Record: This study enrolls outpatients who are 
receiving a treatment intervention that may lead to physical or psychological adverse events 
and will be using the phlebotomy lab to collect blood for serum progesterone levels and 
pregnancy tests, which will be identified by the subjects VA identifiers. Because this study will 
request a Certificate of Confidentiality, a VHA health record will not be created. Instead a note 
entry will indicate that an individual has been enrolled in a research study, any details that 
would affect the subject’s clinical care, and the name and contact information for the 
investigator conducting the study. Subject’s informed consent forms and HIPPA authorization 
documents are not to be included in the health record. No pharmacologic agents are being 
given as part of the study, no research results being used for clinical care, the study 
interventions include a yoga or walking intervention, psychological forms and assessment 
instruments, electrocardiogram data, and imaging studies at the McLean Hospital. 
 
4.5.2.7. 
Creation of a McLean Health Record: The McLean Hospital will create a medical 
record for the anatomic scan in which the subject will be identified by their name, date of birth, 
address, telephone number and social security number. This is a McLean Hospital policy and 
they will obtain the required HIPPA authorization for this record. This record will be kept 
separate from the subject’s research information in which they will be identified by their study 

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identification number. 
 
4.5.2.8. 
Enrollment: Participants are considered enrolled in the study when they give 
oral consent to the telephone interview. The Project Manager and research staff will 
usually do the telephone- 

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interview consent and telephone interview. Participants are considered enrolled in screening after 
they sign the written informed consent. The written informed consent can be obtained by research 
staff, however, the study MD needs to consent the subject with regards to the risks of the imaging 
part of the study prior to scanning. There are two lines on the IC for staff, such that the research 
staff can start the evaluation without the study M.D. Participants are considered randomized when 
they participate in their first intervention. This study uses walking as an active control. There is 
considerable literature to support the use of exercise as a treatment for depression, such that there 
is no deception in this study. 
 
4.5.2.9. 
Recruitment: This proposal contains a randomized controlled trial in a clinical 
population in which 70 subjects will complete the protocol and around 420 will have a 
telephone-screening interview. Although this efficacy trial is not large or multi-centered, it is 
considered a Phase 2 trial due to the previous trial in healthy controls (Study 2) and the 
dosing trial in depressed subjects (Study 4). 
Sample size calculations, based on power analyses, require the following number of completers equally 
divided between the yoga and walking groups to test the hypotheses for each dependent variable: n=70 
completers to test Aim 1: Depressive Symptoms and Mood Scale, n=56 to test Aim 2: GABA changes 
and n=68 to test Aim 3: RSA changes. To meet the criteria for each Aim/Dependent Variable 70 
subjects will complete the intervention with 56 having usable MRS data. Using data from Study 4, with 
consideration of changes in inclusion and exclusion criteria and research setting, the following 
recruitment schedule will be required during a 5 year study period, which will include over 4 years of 
recruitment and treatment. The required total recruitment and annual requirements at each stage are as 
follows: telephone screen 420 total or 105/year; screening interview 173 total or 43/year; randomization 
100 total or 25/year; and completers 70 total or 18/year over a 4 year recruitment period. 
 
 
4.6 Risk/Benefit Ratio 
 
4.6.1. Potential Risks and Methods to be Used to Minimize Risks 
 
4.6.1.1. Source Materials: All data collected in this study will be used specifically for research 
purposes. Types of data will include scores from psychological instruments, screening and monitoring 
forms, electrocardiogram (ECG) data and imaging data. To ensure confidentiality, all data collected will 
be marked with subject IDs not the subjects name, this includes paper and electronic collection. An 
exception to this will be serum progesterone and B-HCG levels that will be processed via the VABHCS 
laboratory. A master sheet with individual names and their respective code numbers will be kept in a 
separate locked file or password-protected computer file that can only be accessed by the research 
staff. The information gathered will only be used for scientific, educational, or instructional purposes. 
Only the research team will have access to the records. Exceptions to this rule will be made for 
representatives of the FDA, other U.S. government agencies, and other mandatory bodies as required 
by law. Forms or requests with subject names include 1) the identifiable information clinical reporting 
form (CRF), and 2) signed Informed Consent. These forms and any other form with subject names will 
be kept in a file separate from the other CRFs. 
 
4.6.1.2. Major Depressive Disorder (MDD) Risks: There are a number of controlled treatment studies 
where the practice of yoga and exercise has been associated with decreased depressive symptoms. 
Neither the practice of yoga or exercise in the form of walking has been established as treatments for 
MDD. Subjects may experience increased depressive symptoms during the study. There maybe yoga 
or walking program outside of this study available in the VA or in the community 
 
 4.6.1.3. Magnetic Resonance Imaging (MRI) Risks: Claustrophobia while inside the magnet for the 
magnetic resonance (MR) assessments can potentially precipitate a panic attack in some participants. MR 
procedures do not involve ionizing radiation and therefore pose no significant radiation risk to participants. 

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However, risks do exist for persons who have metallic objects near vital structures, such as pacemakers or 

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aneurysm clips. Individuals with shrapnel from bombs or iron filings in the eye (mostly welders) would be at 
risk as well. We have a detailed MR Safety Screening Form that every person must complete before he/she 
receives an MR scan. The greatest risk associated with a MR exam is during the time the participant is 
escorted into the magnet suite or away from the magnet, as nearly all of the adverse events in MR suites 
have involved a ferrous metal projectile being drawn into the bore and striking the person. Our sites have 
very extensive safety awareness plans in place complete with detailed signage of magnetic field strength. 
All personnel who escort participants or patients into the scanner receive mandatory training and 
certification. In addition, the imaging sites have complete code carts equipped with cardiac monitors, 
defibrillators, suction, oxygen, and a full range of medications for any medical emergency that might occur. 
 
 
4.6.1.4. Psychological Risks: Subjects may find questions about their history distressing or 
uncomfortable. They may feel nervous when answering psychological questions. A member of the 
research team will be with the participant at all times during the study sessions and will be able to 
answer questions concerning procedures and psychological tests. Subjects will be informed (by trained 
study personnel and the informed consent form) that they can decline to answer any questions, decline 
any procedures, or withdraw from the study at any time. 
 
4.6.1.5. Alcohol Related Risks: As subjects will be allowed to have an Alcohol Use Disorder with up to 
three criteria using DSM V criteria, there is a possibility that they would come to the intervention 
intoxicated and could injury themselves due to intoxication. 
 
4.6.1.6. Physical Risks: The inclusion/exclusion criteria are designed to exclude subjects who would 
be at risk from participation in the yoga or walking interventions. Subjects may experience muscle 
stiffness or discomfort from the physical components of the interventions. In Study 2, healthy yoga 
naïve controls were randomized to a yoga or walking intervention. In Study 4, yoga naïve subjects with 
MDD were randomized to two or thee yoga sessions a week. Adverse events were monitored on a 
weekly basis during the 12-week intervention. In Study 2, minimal physical discomfort related to the 
interventions was reported in both the yoga and the walking group, with slightly greater discomfort 
reported in the yoga group. In Study 4, subjects reported muscle soreness associated with the yoga 
intervention (13/30 subjects) that resolved with modifications of the postures. There is minimal risk of 
injury during the recording of the electrocardiogram (ECG) data using the Faros 180 Sensor (Bittium 
USA, Inc, Bothell, WA), a hand held device powered by a 3,7 volt battery. There is some discomfort 
associated with the placement and removal of the electrodes. Some subjects, primarily males due to 
chest hair, find the removal of the ECG electrodes to be uncomfortable. 
 
The YMCA Step Test is a standardized test that has been used for 30 years as part of the YMCA 
Fitness Testing and Assessment Manual. For this study a modified version of the YMCA step test (Step 
Test) is used as the 12-inch step has been reduced to an 8-inch step to make the procedure easier for 
subjects with short stature. In this protocol, the test consists of stepping up and down an 8-inch step in 
time to a 96 per second metronome. There are four beats to each cycle (step up foot 1, step up foot 2, 
step down foot 1, step down foot 2) for a total of 26 step cycles per minute. Subjects will do the YMCA 
Step Test for three minutes with a 6-minute rest before and after. The YMCA Step test will be done 7 
times during the protocol. Subjects could find the Step-Test to be physically challenging or difficult to 
preform safely. 
 
4.6.1.7. Confidentiality Risks: The yoga and walking interventions are conducted in groups. While 
subjects are informed that what occurs in the group is confidential, there is no guarantee that a 
participant may say something outside of the group that does not respect the confidentiality of the 
group. While source documents are marked with a subject code, there is a risk of a loss of 
confidentiality. Participant could be seen coming to the sessions and a person could deduce that the 
participant is depressed. There is a risk that the electronic data will not be secure. 

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4.6.1.8. Data Transfer Between Collaborating Sites: All procedures involving subjects will take place 
at the VABHCS. Data management and statistical analysis will be done through a contract with the 
Boston University School of Public Health Biostatistics, Epidemiology, Data Analytics Center and or by 
the VABHCS staff a Bedford VA statistician, both have worked on other VA sponsored research 
projects and are familiar with VA procedures regarding the handling of data. VA regulations regarding 
the transfer of data will be followed. 
 
4.6.1.8.1 COVID-19 Risk: The COVID-19 pandemic presents novel risk of infection to both the 
subjects and research staff. A detailed response to this risk has been approved by the VABHCS IRB. 
 
 
PROTECTION AGAINST RISKS 
 
4.6.1.9. Recruitment and Informed Consent Risk Protection: Documentation will be provided to the 
VABHCS that the study personnel have up to date and the appropriate training in the following areas; 
Collaborative Institutional Training Initiative (CITI) for the ethical principals of human research, training 
in Good Clinical Practices (GCP), Privacy, Cybersecurity, and VA Research Data Security and Privacy. 
The research team will also receive training about the HIPAA regulations. This study will include 
children in the 18 to 21-year-old range who will be able to understand and complete the informed 
consent used for all subjects. The consent process will be done in a private space to insure participant 
confidentiality. During the consent process, all subjects will be informed that this is a research study 
and the use of yoga or walking for the treatment of Major Depressive Disorder (MDD) are not a 
conventional forms of treatment. Subjects will be informed that conventional treatments for MDD are 
available, such as psychotherapy, pharmacotherapy, and somatic therapies (e.g., electroconvulsive 
therapy, transcranial magnetic stimulation). Subjects are informed that they can change their mind 
about wanting to have conventional treatment at any time and that the treatment of their depression is 
more important than the completion of the study. Treatment options will be discussed if subjects 
decide not to participate in the study. The Principal Investigator’s primary clinical responsibilities are at 
the VABHCS. In order to avoid confusion between what is treatment and what is clinical care, the 
informed consent will address the issue of therapeutic misconception suchthat the difference between 
clinical care and research will be clearly defined and the subjects will be informed that if they decline to 
participate in the study, there will be no effect on the clinical care that they receive. REDCap, a secure 
web application for building and managing online surveys and databases, will be used to manage data. 
 
4.6.1.10. Risk of Depression: This is a treatment study with modalities that are not approved for the 
treatment of depression. There are alternatives to participation, such as pharmacotherapy, 
psychotherapy or electroconvulsive, and subjects will be informed of these other options and of their 
right to withdraw from the study to seek conventional treatment. Subjects are screened for risk of 
suicide during the screening interview with the Columbia Suicide Safety Rating (C-SSR), a widely used 
instrument to assess suicide risk. It is noted that the C-SSR does not provide a score stating that the 
assessment of suicide is a clinical decision. For this reason, other than the development of suicidal 
ideation with intent, the decision to withdraw a subject remains a clinical matter that is informed by the 
assessments built into the study. There are assessments by a psychiatrist or member of the research 
staff licensed to assess for suicide risk at screening, and at the weeks 4, 8, and 12 assessments using 
the C-SSR. Safety Data from Study 4 showed that out of 30 subjects, 9 out of 10 subjects that 
endorsed suicidal ideation without intent at screening no longer endorsed suicidal ideation without 
intent at the end of the study. No subject developed suicidal ideation with or without intent during the 
study. Suicidal ideation with intent with in a year of screening was exclusionary. In addition, the C-SSR 
evaluation were in agreement with reports of self harm on the Beck’s Depression Inventory II (BDI-II), 
consistent with both clinical evaluation and self-report with regards to safety being accurate. In the 
proposed study, staff will check question 9 of the BDI-II regarding self-harm before the participant 

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leaves the evaluation. Participants will also have a C-SSR at screening and week 4, 8, and 12. The 
screening, week 4, 8, and 12 evaluations are done by research staff defined as being trained and 
certified to assess for safety to self and others (e.g., MD, PhD, LICSW, Master’s level therapist). In 
addition to the monthly evaluations, subjects also fill out weekly adverse event sheets that ask if they 
symptoms of depression are worse or if they want to harm themselves. If increasing symptoms of 
depression are endorsed, this will trigger an evaluation by a research staff or the PI. Some fluctuations 
in depressive symptoms are expected and are not reported as adverse events unless there is a change 
in the level of severity as recorded in the adverse event clinical reporting forms. 
 
4.6.1.11: MRI Risk Protection: We have a detailed MR Safety Screening Form that every person 
must complete before he/she receives a MR scan. All participants with ferrous metal near vital 
structures will be excluded from participating. At the time of the scan, participants will be asked to 
remove all metal objects from their person, including watches, jewelry, change, wallets, credit 
cards, and shoes, which will be kept by the research team in a safe location. Participants are also 
checked with a metal detector prior to entering the scanner. The FDA has determined that MRI 
machines of 4.0 or less do not represent significant radiation risk. Radio Frequency (RF) power 
limits will conform to the FDA guidelines defined in IEF 60601-2-33, the international standard for 
the safety of magnetic resonance devices intended for medical diagnosis. Participants are offered 
earplugs and a set of earphones to reduce the noise level. Blankets and pillows are used to make 
the subject more comfortable. There is a two-way audio system in the scanner through which the 
research team and the participant can communicate at all times. Although the experience of being 
in the scanner can be uncomfortable, it is our experience that a majority of subjects tolerate the 
procedure quite well. Subjects with clinically significant claustrophobia will be excluded from this 
study. 
 
4.6.1.11. 
4.6.1.12. Psychological Risks Protection: A member of the research team will be available to answer 
questions concerning procedures and psychological tests at all times during the study sessions. The 
practice of yoga or walking interventions is unlikely to cause adverse psychological reactions. The 
exception is that subjects with a history of trauma may find the yoga postures elicit memories of the 
trauma. All subjects are screened for trauma and those with a history of PTSD or PTSD symptoms are 
monitored for increasing symptoms during the study. The yoga instructors will be instructed on methods 
for addressing the special needs of trauma victims. Subject with dissociation due to PTSD could be 
excluded at the discretion of the Principal Investigator. In the unlikely event that a subject has an 
adverse experience during an intervention session, clinical staff in addition to the yoga instructors will 
be available during the interventions to assess subjects who are in distress. The inclusion criteria are 
designed to identify subjects with MDD who are not a danger to themselves or others and who are a 
low risk to have an adverse reaction to the interventions. As participants are Veterans and entitle to 
care form the VA, if there is a risk of harm to self that would require hospitalization, they will be taken to 
the walk in clinic or the ER for further evaluation. 
 
4.6.1.13. Alcohol Risk Protection: In order to prevent subjects under the influence of alcohol from 
participating in evaluations or the interventions, all subjects will have a Breath Alcohol Concentration 
(BAC) done after the written informed consent and before participation in the weeks 4, 8, and 12 
evaluations and at any evaluation/intervention if research staff are concerned a participant is under the 
influence of alcohol. If the BAC reading is not zero, participants will not be able to participate in the 
evaluation/intervention. If the BAC is above 0.08% or 0.08 mcrg/l, the level of legal intoxication in 
Massachusetts, the participant’s car keys will be taken until their BAC is less that 0.08 %. The BAC will 
be done with a hand held Alcosensor machine Subjects will be assessed by clinical staff for safety prior 
to leaving if their BAC was greater than zero. These procedures are outlined in the informed consent 
such that participants are aware of these procedures prior to signing the informed consent. 

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4.6.1.14. Physical Risks Protection: To facilitate the evaluation of a subjects ability to safety comply 
with the interventions, they will as part of the inform consent process allow study staff to review their 

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VA medical records as needed. If there is a question concerning a medical problem that would be a 
contraindication to participation that is not in their VA medical record, the Veteran will be asked to sign 
a release of information to allow contact with their medical care clinicians to resolve the issue. If the 
Veteran declines, they maybe excluded from the study. All subjects will be assessed by a staff 
physician for a history of prior injuries or medical conditions that would preclude the subjects from safe 
participation in the yoga intervention. All subjects will have a physical examination at the screening visit 
as part of their safety assessment. The yoga intervention for the proposed study was piloted in Study 2 
and 4 and was associated with minimal adverse events. This study uses certified yoga Instructors with 
Iyengar training and at least 5 years of teaching experience. Iyengar yoga is designed so that 
adaptations are made on an individual basis using position modifications and props such as blocks, 
blankets and belts. This individualized approach allows physical limitations of the subjects to be 
addressed and reduces and/or minimizes the risk of injury and muscle soreness. The Faros Bittium will 
be used in accordance with the manual. Staff that has been trained to perform the procedure will do 
the placement and removal of the electrodes. The Principal Investigator who is boarded in neurology 
and used to work at a Rehabilitation Hospital will screen subjects for difficulty with the Step Test prior 
to randomization. 
 
4.6.1.15. Protection of Special Populations: Individuals with psychiatric illness are considered 
special populations. This study will enroll subjects with MDD without psychomotor depression, 
psychosis, or agitation that would prevent participation in the study. This level of depression should not 
affect cognition to the degree that they would be unable to consent to the study. Psychiatric disorders 
that would impair the ability of subjects to consent such as bipolar disorder, psychosis and dementia 
are exclusionary. All subjects take a quiz after signing the consent to assess if they understand the 
risks and benefits of the study. A subject may be excluded if he/she make errors on the quiz and cannot 
demonstrate that he/she understands the error. Yoga is safe for use in pregnant females; in fact yoga 
is being studied as a potential treatment for perinatal depression. Accordingly there is minimal risk to a 
pregnant participant or the fetus from participation in this study. However the presence of pregnancy 
would be a confounding variable as the risk of depression can increase during pregnancy. For the 
integrity of the study, pregnant females are excluded. In order to avoid unnecessary procedures, 
females will be asked if they are pregnant or intend to become pregnant and are required to use an 
acceptable form of birth control during the study. Only if there is a change in their normal menstrual 
cycle such that pregnancy is a concern will females be considered for exclusion. All females of child- 
bearing capacity must use one of the following approved methods of birth control: oral contraceptives 
("the pill"), contraceptive implants under the skin, contraceptive rings or patches or injections, 
diaphragms with spermicide and condoms with foam, surgical sterilization, or complete abstinence from 
sexual intercourse. IUDs with progesterone implants are not allowed because they can cause 
anovulation. IUDs with metal are not allowed due to the MRI scans. 
 
4.6.1.16. Confidentiality Risk Protection: Information is kept in separate databases. 1) To ensure 
confidentiality, all information on clinical report forms (CRFs) or placed directly into REDCap will be 
marked with subject IDs so that it cannot be associated with any individual. 2) A master list, with 
individual names and their subject IDs, will be kept in a locked file cabinet or password-protected 
computer file that can be accessed only by research staff. 3) Identifiable information is kept in a third 
database. The information gathered will only be used for scientific, educational, or instructional 
purposes. Exceptions to this rule will be made for representatives of the FDA, other U.S. government 
agencies, and other mandatory bodies as required by law. Because of the sensitive nature of the 
questions surrounding the use of illegal substances, a Certificate of Confidentiality will be obtained. 
While subjects are instructed that the groups are confidential, by the nature of conducting the classes 
in a group the confidentiality of what is said in the group cannot be guaranteed. For this reason, 
subjects are instructed to monitor what they say in the group settings. Subjects are informed that they 
can ask to speak to a staff member in private. 
 
4.6.1.17. COVID-19 Risk Protection: In addition to the already approved safety measures for when 

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the participants are at the VABHCS additional measures to increase social distancing have been 
added. The change of the in-person interventions to virtual interventions except for the randomization, 
and weeks 4, 8, and 12 evaluation is a change that could affect the scientific integrity of the study. As 
no subject had started the interventions prior to the shutdown of all non-essential research in March 
2020, all subjects will have the same experience of virtual interventions. The Veterans Administration 
Central Office (VACO) Project Manager has given tacit approval to this change, provided it is IRB 
approved and the documents reflecting VACO approval of the changes are filed by the VABHCS 
ACOS with VACO. The changes to the intervention to make them virtual have been listed in the 
protocol. The video connections will be made with VA Video Connect or another platform approved by 
the VABHCS Information System Security Officer (ISSO). 
 
4.6.2. Data Safety Monitoring 
4.6.2.1. Data Monitoring Committee (DMC): Clinical Science Research and Development (CSR&D) 
has a centralized Data Monitoring Committee (DMC) that monitors clinical studies that are approved for 
funding, include human subjects.  The CSR&D DMC provides an ongoing independent evaluation of 
the progress of studies, including participant accrual and retention, adverse events monitoring, and 
analysis plan. Loss or theft of data is considered an adverse event that would be reported to the DMC. 
The DMC is a service that is provided by CSR&D to ensure independent oversight of the safety and 
integrity of the project. No other DMC or Data and Safety Monitoring Board (DSMB) review is needed. 
The DMC office is located within the Cooperative Studies Program Coordinating Center (CSPCC) at the 
Edward Hines, Jr. VA Hospital in Hines, IL. 
 
4.6.2.2. Suspension of the Study and Breaking the Blind: The DMC that has the authority to 
suggest suspension of the study if subject safety is at risk. The final decision regarding suspension lies 
with VACO. Because the subjects are not blinded to the yoga or walking intervention they are receiving, 
a protocol to break the blind concerning group assignment is not required. As was done in March 2020, 
the study maybe suspended due to an order from VACO related to the COVID-19 pandemic. 
 
4.6.2.3. Data Storage and Period of Data Retention: Hard copies of research data will be kept 
according to VA regulations. Computer Files will be locked and maintained according to hospital and 
research standards. All computer data will be stored on a firewall-protected server. Computer data will 
be password-protected and access will be restricted to research staff and the Principal Investigator. 
The informed consents have a section giving permission for the data to be used "to study the effects of 
yoga on disease, health and well-being” such that analysis of the data that falls under this description 
has already been agreed to by subjects. The approved IC already documents that if a data repository is 
created in the future subject consent has been obtained. If subjects do not check this box, their data will 
not be used for to additional analysis. Currently, VA regulations require that data be retained for 5 years 
after the end of the fiscal year in which the project is closed at which time the data will either be 
destroyed or put into a repository. Permission to create a data repository that combines the data from 
the Merit Award, the R01 and R21 on which the Merit Award is based is requested. 
 
4.6.2.4. Compliance with VA Regulations: The REDCap database and the final dataset will be a 
Limited Data Set that contains only the date of data collection. HIPPA identifiers include the data of 
data collection, but these dates it will not allow reasonable identification of the subjects. 
The Azure Rights Management Services (RMS), system that has been approved for use by VA 
researchers to send information to both subjects and other researchers. After the original contact, potential 
participants will be given an option to communicate via VA approved secure email system designed for 
patients/participants use. Besides contact information and dates of service, no personal health information 
will be released by email. Subjects will be instructed in how to use Azure to send and receive data. Azure 

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RMS will also be used to transfer a blinded dataset of electrocardiogram data to Dr. Heilman in the 
University of North Carolina for analysis. The only Private Health Information (PHI) that will be transferred 
is this limited data set are the dates of the intervention. A user guide will be provided to Dr. Heilman 
detailing the proper procedures for replying to and sending data as an Azure RMS protected message. 
4.6.2.5. Protection of Data: Study data will be collected using the of REDCap data collection system, a 
software tool developed at Vanderbilt University and made available through the Clinical and 
Translational Science Awards network (CTSAs). To help protect and secure the data stored in 
REDCap’s database, the software application employs several methods to protect against malicious 
users who may attempt to identify and exploit any security vulnerabilities in the system. Access to the 
REDCap data entry website will be based on permissions granted by username and password. Only 
authorized study members will be able to enter or view data. The login information (username) of the 
person submitting the information, the date and time submitted, and other navigational information will 
be automatically obtained and stored in the database. When available, staff will sign subjects into a 
secure link to access REDCap, such that subjects will have access only to required assessment forms 
that they will fill out in REDCap:) Beck Depression Inventory (BDI-II), Spielberg State-Trait Anxiety 
Inventory (STAI), PTSD Check List – Civilian (PCL-C), Exercise-Induced Feeling Inventory (EIFI) and 
Pittsburgh Sleep Quality Index (PSQI). 
 
4.6.2.6: Data on Hard Drives: It is anticipated that the study will be provided with three computers.. 
These computers will be encrypted. It is anticipated that the study staff will be provided with mobile 
devices, one for the PI, one for the project manager and and one for the clinical staff, these devices will 
comply with encryption technology that is FIPS 140-2 validated. 
 
4.6.3. Potential Benefits 
 
4.6.3.1. General Benefits: All subjects will have the benefit of an evaluation of their depressive 
symptoms by clinical staff. Subjects, who have a MRI scan at the McLean Hospital, will have the 
advantage of having the anatomic study read as part of the safety protocol. 
 
4.6.3.2. Benefits of the Yoga Intervention: The yoga intervention in this protocol includes yoga 
postures and yoga breathing. Both practices have been shown to have health benefits in controlled 
trials of normal subjects and clinical populations. In addition to the benefit of physical activity, the yoga 
intervention includes exercises for flexibility, strength and balance. Yoga practices have been 
associated with stress reduction, weight reduction, improved sleep, and decreased blood pressure to 
name a few of it's health benefits. Yoga practices have been shown to decrease symptoms in disorders 
with low GABA activity such as depression, post traumatic stress disorder (PTSD), epilepsy and chronic 
pain. There have been controlled studies showing yoga-based interventions to be effective in treating 
depression ranging from mild depression symptoms to major depressive disorder. The benefits to the 
subjects participating in this study include the both the general health benefits of yoga and the potential 
to treat the depressive symptoms. 
 
4.6.3.3. Benefits of the Walking Intervention: There is an extensive literature that exercise improves 
mood such that the walking intervention is an active control. The walking intervention includes the 
benefit of regular exercise. 
 
4.6.3.4. Additional Benefits: Social interaction with the research staff and the group members in a 
population that can be socially isolated is a benefit. Additional benefits include an evaluation by a 
physician who will discuss treatment options if the subject does not participate in the study. Subjects 
are closely monitored during the study for increasing symptoms of depression. 
 
IMPORTANCE OF KNOWLEDGE TO BE GAINED 

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4.6.3.5. Understanding the Relationship Between Yoga Practices, Parasympathetic Tone and 
Mood: Integrated medical practices such as yoga and exercise are becoming increasingly popular. It is 
critical that the mechanisms through which a yoga practice confers benefits in the form of reduced 
stress and improved affective states be elucidated. This study will examine the relationship between 
yoga, the activity of the parasympathetic nervous system and mood. Decreased parasympathetic tone 
has been documented in a number of psychiatric diseases, with low high frequency heart rate variability 
(HRV), a marker of low parasympathetic tone being a marker for overall mortality. If this study confirms 
the findings in the Preliminary Data Section that a yoga intervention is associated with increased 
parasympathetic tone and improved mood in a Veteran population, it would have significant 
implications for treatment of disorders with low parasympathetic tone such as depression, PTSD, and 
Alcohol Use Disorder, all disorders with a high healthcare burden in the Veteran population. Reduction 
in Suicidal Ideation: There is currently significant concern about the rate of suicide in the Veteran 
population is higher than the civilian population. The proposed intervention resulted in the elimination of 
suicidal ideation without intent present at screening in a prior dosing study in 9 out of 10 subjects, or by 
approximately 30% in the population that completed the study (9/30). 
 
4.6.3.6. Use of Yoga Intervention as Monotherapy or as an Adjunct for Partial Remission: The 
development of an intervention that can be used as monotherapy and as an adjunct to other treatments 
for depression that has the documented ability to decrease suicidal ideation would be an advance for a 
field in which up to 40% of those treated do not achieve remission. The Preliminary Data Section shows 
improvement of depressive symptoms in subjects on a stable dose of monoamine therapy when the 
proposed yoga intervention was added to their treatment. A better understanding of the 
neurophysiology underlying the practice of yoga could advance the treatment of many disease 
processes, as well as reduce the costs to the afflicted individuals, the health care system, and society 
 
4.6.4. Alternative Procedures 
 
The alternative is to not participate in the study. Participants may choose to seek standard care for their 
depression such as psychotherapy, medications electroconvulsive therapy (ECT), and transmagnetic 
stimulation (TMS). 
 
4.7 
Costs and Payments 
 
Subjects will be paid for their time: $10 for each group session, $20 for the week 4 evaluations, $40 for 
the week 8 evaluations, $60 for the week 12 evaluations, and $40 for each MRS scan for a maximum of 
$480. Payments are made one or twice during the study, depending on the participants request via 
check. Subjects are not required to pay for services outside of the VA with the exception as to whether 
they choose to drive to the McLean Hospital, were parking is free or decide to take public transportation 
to the McLean Hospital. If a subject provides proof of the cost for transportation (e.g., taxi, Uber, or 
Lyft) on the day he/she has a scanning session, the participant will be compensated for his/her the cost 
of travel at a rate of $40 for each leg of the journey, or $80 round trip. If participants decide to keep the 
yoga blocks after the intervention, $20 will be deducted from their payment. Subjects are paid for the 
scans at the McLean Hospital as long as they are compliant with the protocol (e.g., As they are 
instructed not to drink prior to the scan in the informed consent, if they arrived for the scan under the 
influence of alcohol they would not be paid. If there were a scan malfunction, such that the subject 
arrived and could not be scanned, they would be paid for one scan. If they agreed to come back they 
would also be paid for that scan). 
 
4.8 
Providing for Reuse of Data: 
 
The data will be also be stored in the Boston Yoga Research Center (BYReC) data repository being set 

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up here at the VABHCS, for future research studies pertaining for future research studies pertaining to 
the effects of yoga and walking on disease, health, and well being. All data will be stored and 
maintained according to VA regulations and only investigators approved through the Social and 
Community Reintegration data repository committee will have access to this data. Any future use of the 
data will be reviewed and approved by this committee. The creation and management of this data 
repository will be approved by the hospital IRB and Research and Development Committee before any 
of the data from this study will be stored in it for future use. Once the Data Repository has been 
approved, an amendment to this project will be submitted to store the data in the data repository. 
 
4.9 Creation of a Tissue Bank: No Tissue Bank will be created 
5.0 RESOURCES 
 
This is a research study such that all evaluations, interventions, intervention assessment and laboratory 
testing for serum progesterone and pregnancy test will be done as part of this research study. The 
imaging component of this study will be done via a contract at the McLean Hospital. This study is 
staffed for the PI to have a 3/5th time position with the VA. There is a full time project manager and a 
50% time clinical staff at the ENRVA Hospital. Dr. Chun Zuo, a Co-Investigator with an appointment at 
the McLean Hospital, will oversee imaging acquisition and the analysis of the spectral data. Dr. Keri 
Heilman is an expert in analysis of ECG readings for analysis of RSA. Dr. Richard Brown and Dr. 
Patricia Gerbarg are experts in the use of alternative treatments for psychiatric disease and will serve 
as consultants is there are clinical concerns regarding the patients. 
 
Dr. Streeter has the following space in Bldg. 70, two offices, 119 and B31, the use of a large room for 
the in-person yoga intervention and a large hall with low traffic for the in-person walking interventionThe 
clinical staff will use one of the offices on the first floor for the week 4, 8, and 12 assessments, while the 
interventions will take place on the basement level, this separation will decrease the likelihood that 
clinical staff will be exposed to subjects during the intervention such that they would no longer be 
blinded to the interventions. 
 
A. BEDFORD VETERANS ADMINSTRATION MEDICAL CENTER 
 
Bedford Veterans Administration Medical Center (VAMC) serves a large number of Veterans with 
mental illness who are and are not in treatment and thus provides a clinical population that would both 
benefit from the proposed study and will allow recruitment of the require research subjects. The 
resources below outline the extensive services and resources available at the Bedford VAMC. 
 
I. CLINICAL RESOURCES 
 
Inpatient Psychiatry: Bedford has an acute inpatient psychiatry unit (30 beds) as well as a sub- acute 
unit (22 beds). Both programs have incorporated a recovery philosophy that orients Veterans to the 
goal of community integration as eventual outcome for all treatment. Peer specialists and community- 
based activities have been incorporated into the programs over the last several years. 
 
Outpatient Mental Health Clinic: The outpatient clinic serves 5,000 Veterans per year, providing the full 
range of outpatient mental health services. In 2015, there were 3,971 patients receiving treatment with 
antidepressant medications. The clinic staff is organized into five interdisciplinary teams consistent with 
the Behavioral Health Interdisciplinary Program (BHIP) model, and has served as an exemplar of the 
BHIP program. The Mental Health Clinic provides services through three Community Based Outpatient 
Clinic (CBOC)’s in Gloucester, Haverhill, and Lynn, Massachusetts. 
 
Specialty Treatment Programming for Substance Use Disorders: The Veterans Mental Health and 

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Addictions Program (VMHAP) is an intensive day treatment program for substance use disorders. It serves 
500 Veterans per year. The Aftercare Program provides a range of outpatient services for substance use 
disorders to 300 Veterans per year. 
 
Integrated Mental Health and Primary Care Program: The Integrated Mental Health/Primary Care 
Program uses a co-located model of care to support the provision of mental health services by primary 
care providers and to facilitate the transition of some Veterans to specialty mental health care. 
 
II. RESEARCH 
 
Previous Research: The Outpatient Mental Health Clinic has been the site of numerous prior research 
studies with excellent staff support and interest. Both in-patient units have been the sites for prior 
research studies. There is a history of meditation and yoga-based research studies being conducted at 
the Bedford VAMC with high interest from the patient population. 
 
The Social and Community Reintegration Research (SoCRR) is a VA Rehabilitation Research and 
Development (RR&D) Reserve Educational Assistance Program (REAP) located at Bedford, bringing 
together a diverse group of VA researchers as a consortium to address the urgent need to build 
capacity for high quality research in the priority area of community reintegration for Veterans with 
psychiatric disabilities. Veterans with psychiatric disabilities are often withdrawn from or are at risk for 
having difficulty sustaining community involvement because of the effects of their psychiatric 
disabilities. SoCCR’s focus on critical areas of community reintegration, including employment, 
education, and interpersonal relationships, converge on the central mission of helping Veterans 
become more engaged in needed treatment that targets reasons for poor community involvement (e.g., 
difficulty maintaining jobs or education, relationship difficulties, low social support). 
 
The New England Mental Illness Research Education and Clinical Center (MIRECC) is co-located at 
the Bedford and West Haven VA’s. The New England MIRECC focuses on rehabilitation research and 
educational services designed to address the needs of Veterans with combined mental illness and drug 
or alcohol dependence (dual diagnosis). The aim of the MIRECC is to improve the treatment of dually- 
diagnosed Veterans by developing innovative new treatment for co-morbid substance use disorders, 
devising more effective ways to deliver existing treatments, and creating better programs to train VA 
treatment providers in therapies with proven efficacy. The research program is comprised of an 
interrelated series of studies at three levels: a) health services research, b) applied clinical research 
and c) basic clinical research on the neurobiological basis of dual diagnosis. 
 
The Center for Healthcare Organization and Implementation Research (CHOIR) is co-located at the 
Bedford and Boston VA Medical Centers, and is one of the 19 Centers of Excellence within the 
Veterans Administration Health Services Research and Development Program. The Center's mission 
is to improve Veterans' health outcomes by developing, studying and applying evidence-based 
practices that will be widely implemented and sustained. The three research priority areas are: 
Recovery in behaviorally vulnerable populations, Medication optimization, and Public health 
communication. 
 
The National Center on Homelessness Among Veterans: With researchers located at the Bedford and 
VA Connecticut Healthcare System campuses, this Center seeks to promote recovery-oriented care for 
Veterans who are homeless or at risk for homelessness. The Center is a national resource that 
conducts research to inform practices tied to the VA five-year plan to end homelessness among 
Veterans. The Center has four resource cores: Policy Analysis, Model Development & Implementation, 
Education & Dissemination, and Research & Methodology. The Boston University Center for 
Psychiatric Rehabilitation, located in Boston, Massachusetts, has been primarily a training collaborator 
with staff at Bedford, and has assisted VACO in guiding VA psychiatric rehabilitation services 
development. The Center is affiliated with the Sargent College of Health and Rehabilitation Sciences at 

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Boston University. The Center is a research, training, and service organization dedicated to improving 
the lives of persons who have psychiatric disabilities by improving the effectiveness of people, 
programs, and service systems. Its work is guided by the most basic of rehabilitation values that first 
and foremost, persons with psychiatric disabilities have the same goals and dreams as any other 
person. They want a decent place to live, suitable work, social activities, and friends to who to they can 
turn in times of crisis. The mission of the Center is to increase knowledge in the field of psychiatric 
rehabilitation and to apply this body of knowledge to train treatment personnel, to develop effective 
rehabilitation programs, and to assist in organizing both personnel and programs into efficient and 
coordinated service delivery systems. 
 
The Boston University School of Medicine (BUSM), Department of Psychiatry, located in Boston 
Massachusetts is the primary academic affiliation for the Bedford VAMC. Many of the Bedford VAMC 
psychiatry staff has academic appointments at the BUSM. The department’s residents and addiction 
fellows rotate through the Bedford VAMC inpatient units and outpatient clinics. This affiliation provides 
additional support for research endeavors at the Bedford VAMC. 
 
III. PROGRAMS FOR VETERANS RECEIVING MENTAL HEALTHCARE 
 
The following programs are potential sources for recruitment. 
 
The Community Reintegration Program is a national exemplar, designed specifically to help Veterans 
currently engaged in mental health care at the Bedford VAMC to become more fully engaged in 
community activities. The program is staffed by four full-time peer specialists and one psychologist. 
Each peer is assigned to a geographical area where many of the Veterans served at Bedford either live 
or want to live once treatment is complete. Each peer has developed relationships with community 
stakeholders and resources in key categories: (1) special Veteran groups (peer support groups, 
Veterans of Foreign Wars (VFW), Disabled American Veterans (DAV), etc.); (2) other peer support 
groups (non-Veteran peer support groups, Alcoholics Anonymous (AA), Narcotic Anonymous (NA), 
etc.); (3) local Veteran-specific programming (town Veteran Services Officers, Veteran housing and 
employment specialists, etc.); (4) recreation groups (local softball leagues, etc.); (5) religious and 
spiritual communities, including those with significant numbers of Veteran members; and (6) individual 
Veterans in the community who are willing to support and mentor Veterans transitioning out of care. 
 
Bedford Vocational Services: The Bedford Vocational Services program (also called Compensated 
Work Therapy or CWT) has been one of the largest VA vocational programs in the country for many 
years, and has been a national leader in innovative programming. Supported Employment (SE), 
Transitional Work Experience (TWE), and Incentive Therapy (IT) are all vocational rehabilitation 
programs supported at the Bedford Campus. Over 300 Veterans are admitted to the program each 
year. Approximately 150 Veterans receive Supported Employment per year, making Bedford one of the 
largest VA providers of Institute of Psychological Services (IPS) Supported Employment. 
Approximately 130 Veterans participate in Transitional Work Experience, the VA’s traditional model of 
vocational services. These Veterans work in the medical center and in the community while they 
prepare for returning to competitive employment. Supported Self Employment is an adaptation of IPS 
Supported Employment, designed specifically for Veterans interested in self-employment. The program 
serves 50 Veterans per year and has been now successfully disseminated to the West Haven VA, 
Boston VA, Manchester and Brockton VA’s. The Supported Education program is one of less than 10 in 
the VA nationwide, and represents a new intervention designed to serve the education needs of 
returning Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF) Veterans. 

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Peer Services: Certified peer specialists represent a key resource for encouraging community 
integration/reintegration, as they model the successful return of Veterans with mental health problems 
to full community involvement. Bedford’s program has been identified by VACO as a national 
exemplar, with 13 full-time Certified Peer Specialists working in virtually every mental health program 
and in Primary Care. Peer specialists provide a range of services, including introducing Veterans to 
programs, helping them navigate the VA system, providing informal support, modeling successful 
functioning, and running peer support groups. Strategically, they are among the most important staff 
during the transition from active engagement in treatment to full community re-integration. The Bedford 
Peer Services Program is highly involved in the VACO peer services program and offers education and 
training to peer specialists from around the country through the Mental Illness Research Education 
Clinical, Centers of Excellence (MIRECC) Peer Education Center. 
 
The Safing Center is a specialty outpatient mental health program that provides consultation, crisis 
management, and individual and couple therapy to Veterans who are using and/or experiencing 
violence in their intimate relationships. Additionally, the Safing Center provides outreach, consultation, 
and education and training, services to staff and Veterans, locally, regionally, and nationally. The Safing 
Center approaches relationship violence from a comprehensive, Veteran-centered, recovery-oriented 
framework, which includes attention to all domains of health (physical and mental health, relational 
health, occupational/educational health). The Director of the Safing Center has pioneered new 
interventions for (1) identifying and (2) treating Veterans who are using and/or experiencing violence in 
their intimate relationships. The Safing Center has been designated as a national exemplar by VACO, 
and interventions developed at the Safing Center are being disseminated by VACO to 5 other pilot 
sites. 
 
Other VA Mental Health Rehabilitation and Recovery Programs: Bedford has been a national leader in 
developing recovery-oriented rehabilitation programs for Veterans with mental illness. Bedford has 
some of the largest rehabilitation services in the nation including one of the largest vocational 
programs, the largest Transitional Residence (serves 80 Veterans per year), one of the largest Mental 
Health Intensive Case Management (MHICM)’s (serves 120 Veterans per year), and one of the largest 
Veterans Administration Supportive Housing (VASH) programs (serves 200 Veterans per year). 
Virtually all of these programs have been sites for prior research studies and staff has been good 
partners in VA research efforts. The MHICM program has been a national leader in Acceptance and 
Commitment Therapy (ACT) programming supported by relationships with mental health professionals 
and has been designated a national center of excellence. The Veterans Community Care Center is a 
Psychosocial Residential Rehabilitation Treatment Program (PRRTP) program designed to help 
Veterans with Serious Mental Illness (SMI) who are living in the community, expand their independent 
functioning in that community. The Domiciliary Care Program is a 50-bed Residential Rehabilitation 
Treatment Program (RRTP) program built around the goal of helping homeless Veterans struggling with 
mental illness and addiction, to rebuild their lives in a community of their choice. The program works 
closely with Vocational Services and the Community Reintegration Program to help Veterans re- 
establish full independence in the community. The VASH program was a pioneer in developing the 
VASH model of homeless intervention, and has been a site for Critical Time Intervention studies. The 
CWT/TR has been the site of studies of efficacy of TR programming and of enhancements like 
contingency management. 
 
Office Space: 
 
Dr. Streeter has the following space in Bldg. 70 to conduct the study, two offices, 119 and B31, the 
use of a large room for the yoga intervention and a large hall with low traffic for the walking 
intervention. There are additional conference rooms that could be scheduled for patient interviews as 
needed. 

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Gymnasium and Pool: 
 
The Bedford VAMC has a large gym, pool and exercise areas that can be used for the interventions. 
 
Computer: 
 
Computer services and equipment will be provided by the VA Information technology office. 
 
6.0 
Collaborations 
There will be a contract with the McLean Hospital for the imaging component of this study. The goods 
and services will include scan acquisition, scan analysis and the associated costs for McLean Staff that 
will include Dr. Zuo, Rose Villefuerte, MRI scan technicians and a to be announced research assistant. 
 
7.0 
Qualifications of the Investigators 
Personal Statement for Key Personnel and Consultants 
Chris Streeter, M.D.: Principal Investigator 
I have worked in the VA system for over 15 years and have extensive experience in treating Veterans 
with psychiatric and neurologic illness. I will have a 3/5th time appointment at the VABHCS when this 
study commences. Either I or the other clinical member of the team to be announced will be 
responsible for the study related healthcare decisions. 
 
For the past 40 years I have pursued studies that explore the mind-body interface. Starting in college 
with Southeast Asia Studies, a neurology residency, a behavioral neurology fellowship and a psychiatry 
residency and as a long time yoga practitioner. I have acquired the skills required to be the Principal 
Investigator for the proposed study. As the Director of Functional Neuroimaging for Psychiatry and the 
Director of the Boston Yoga Research Center, I used magnetic resonance spectroscopy (MRS) to 
measure gamma aminobutyric acid (GABA) in cocaine dependent individuals. I observed that 
depression, anxiety and epilepsy had symptoms that increased with stress and decreased when 
treated with pharmacological agents that increased brain GABA levels, and that yoga interventions also 
improved symptoms in the same disorders. This lead to the hypothesis that yoga in part acted through 
the GABA system, which was refined by the including yoga’s effect on the parasympathetic nervous 
system. This hypothesis was supported by 4 prior studies confirming the association between a yoga 
session, increased parasympathetic tone, increased brain GABA levels and improved mood. I have an 
appointment at the McLean Hospital and have been the PI on previous studies were MRS was used to 
measure brain GABA levels. This Merit proposal extends this research program to include a Phase 2 
Randomized Controlled Trial of the manualized yoga and breathing intervention and walking control 
developed in prior R21 and R01 studies in civilians to Veterans with Major Depressive Disorder I have 
experience in leading multidisciplinary teams and have been a Co-Investigator in nine clinical trials 
sponsored by the National Institute of Health (NIH) and industry. I and my team are devoted to the 
development of rigorous evidence based studies to guide the use of the proposed yoga and breathing 
intervention for the treatment of Veterans with Major Depressive Disorder in the short term with the 
expansion to other disorders and efficiency studies in the long term. The publications listed below 
document previous research in Veterans regarding Post Traumatic Stress Disorder, Alcohol 
Dependence, Traumatic Brain Injury, and all topics important to Veterans and relevant to the proposed 
protocol. 
 
 
Keri Heilman, Ph.D. 
My primary research interests focus on investigating how physiological state influences social 
behavior. I am specifically interested in identifying the neural mechanisms mediating inappropriate 
social behaviors. The long-term goal of my research is to develop tools that could use biobehavioral 
indices as early predicators of psychopathology. I have been a consultant on Dr. Streeter’s Study using 

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yoga to treat depression, where I assisted in the study design with regards to the collection and 
analysis of the Respiratory Sinus Arrhythmia data. I am well qualified to serve as a consultant on the 
continuation of this research in Veterans. 
 
During the course of my research career working with Dr. Stephen W. Porges, I have become proficient 
in the monitoring of physiological indices of social behavior and emotional regulation, such as heart rate 
variability, eye gaze, facial muscle activity, auditory processing and cortisol.  I have been developing 
my specific area of expertise – the collection, editing and analyses of heart rate data for cardiac 
rhythms - since 2000. I have been regularly teaching training workshops on the editing and analyses of 
heart rate data since 2008. 
 
I have experience working with participants of all ages (infants through adult). I have conducted 
research to investigate how difficulties in behavioral state regulation in infants and toddlers (e.g., 
fussiness, crying, tantrums) are related to developmental trajectories. I have additionally worked with a 
variety of clinical populations who share a difficulty in social behavior/emotion regulation, such as 
children with autism spectrum disorder, social anxiety disorder and selective mutism. More recently, I 
have been involved in research studies that evaluate the effects of trauma in children and adolescents, 
and the effects of a biobehavioral intervention on autonomic/behavior regulation and auditory 
processing. 
 
 
REFERENCES 
 
1. 
Wang PS, Simon G, Kessler RC (2003): The economic burden of depression and the cost-effectiveness 
of treatment. Int J Methods Psychiatr Res. 12:22-33.Rush AJ, Fava M, Wisniewski SR, Lavori PW, Trivedi 
MH, Sackeim HA, et al. (2004): Sequenced treatment alternatives to relieve depression (STAR*D): rationale 
and design. Control Clin Trials. 25:119-142. 
2. 
Judd LL, Schettler PJ, Rush AJ, Coryell WH, Fiedorowicz JG, Solomon DA (2015): A new empirical 
definition of major depressive episode recovery and its positive impact on future course of illness. J Clin 
Psychiatry. 
3. 
Fava M, Rush AJ, Wisniewski SR, Nierenberg AA, Alpert JE, McGrath PJ, et al. (2006): A comparison 
of mirtazapine and nortriptyline following two consecutive failed medication treatments for depressed outpatients: 
a STAR*D report. Am J Psychiatry. 163:1161-1172. 
4. 
Porges SW (2007): The polyvagal perspective. Biol Psychol. 74:116-143. 
5. 
Brown RP, Gerbarg PL (2005): Sudarshan Kriya yogic breathing in the treatment of stress, anxiety, and 
depression: part I-neurophysiologic model. J Altern Complement Med. 11:189-201. 
6. 
Brown RP, Gerbarg PL (2005): Sudarshan Kriya Yogic Breathing in the Treatment of Stress, Anxiety, 
and Depression: Part II-Clinical Applications and Guidelines. J Altern Complement Med. 11:711-717. 
7. 
Karavidas MK, Lehrer PM, Vaschillo E, Vaschillo B, Marin H, Buyske S, et al. (2007): Preliminary 
results of an open label study of heart rate variability biofeedback for the treatment of major depression. Appl 
Psychophysiol Biofeedback. 32:19-30. 
8. 
Streeter CC, Gerbarg PL, Saper RB, Ciraulo DA, Brown RP (2012): Effects of yoga on the autonomic 
nervous system, gamma-aminobutyric-acid, and allostasis in epilepsy, depression, and post-traumatic stress 
disorder. Medical Hypotheses. 78:571-579. 
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