HRV Biofeedback in Pain Patients: Pilot Intervention for Pain, Fatigue & Sleep
Phase: NA
Conditions: Chronic Neuromuscular Pain
Sponsor: VA Office of Research and Development

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CLINICAL RESEARCH PROTOCOL 
 
HRV Biofeedback in Pain Patients: Pilot Intervention for Pain, Fatigue & Sleep 
 
 
Principal Investigator:   
James Burch, MS, PhD, Associate Professor 
Health Research Specialist 
WJB Dorn Department of Veterans Affairs 
Medical Center 
6439 Garners Ferry Road 
Building 100, Room 3A156 
Columbia, SC 29209-1639 
Phone: (803) 776-4000 ext. 1-6635 
E-mail: james.burch@va.gov 
and 
Cancer Prevention and Control Program, 
Dept. of Epidemiology and Biostatistics, 
Arnold School of Public Health, University of 
South Carolina, Columbia, SC 
915 Greene St., Columbia, SC  29208 
Phone: 803 576-5659 
E-mail: burch@mailbox.sc.edu 
 
 
Co-Investigator: 
 
Alexander McLain, PhD  
Assistant Professor of Biostatistics 
 
 
 
 
 
 
Department of Epidemiology and Biostatistics  
Arnold School of Public Health  
University of South Carolina, Columbia, SC  29208 
 
Consultants: 
 
Raouf (Ron) Gharbo, DO, PhD 
Adjunct Clinical Faculty  
Eastern Virginia Medical School 
Physical Medicine & Rehabilitation 
Eastern Virginia Medical School  
and 
Staff Physiatrist  
Riverside Medical Group 
Hampton Roads Riverside Regional Medical Center 
Newport News, VA  23601 
 
James  R.  Hebert, ScD 
Health Sciences Distinguished Professor  
 
 
   
Director, South Carolina Statewide Cancer Prevention and Control Program,  
Dept. of Epidemiology and Biostatistics,  
Arnold School of Public Health,  
University of South Carolina, Columbia, SC  29208 
 

 
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Consultants (Continued): 
 
Francis G. Spinale, MS, PhD, MD 
Distinguished Professor of Personalized Medicine 
Professor, Cell Biology Anatomy, and Surgery 
School of Medicine 
University of South Carolina, Columbia, SC  29208 
and  
Staff Physician 
WJB Dorn Department of Veterans Affairs Medical Center 
6439 Garners Ferry Road 
Columbia, SC 29209-1639 
 
 
 
 

 
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PROTOCOL ABSTRACT 
 
Pain initiates a stress response that increases sympathetic output and facilitates autonomic imbalance. Heart  
rate variability (HRV) is a valid, quantitative, noninvasive measure of autonomic function. HRV biofeedback  
(HRV-B) is a novel biobehavioral procedure whereby patients learn to restore autonomic imbalance by  
developing ‘HRV coherence’. Patients in this state have improved mood, cognition, and executive function, and  
HRV-B interventions have been used to treat: pain, anxiety, depression, insomnia and symptoms of 
posttraumatic stress disorder (PTSD) and heart disease. We have found that HRV-B training among Veteran 
PTSD patients can enhance HRV coherence and improve cognition and sleep relative to controls. Importantly, 
our pilot study showed that HRV-B can alleviate chronic pain and stress among Veteran Pain Clinic patients. 
HRV-B thus has a pivotal role in restoring autonomic balance and managing pain and the symptoms that 
amplify it. The proposed project is a randomized, sham-controlled, biobehavioral intervention that will test the 
hypotheses that HRV-B increases HRV coherence and reduces pain, stress, fatigue, insomnia and depression 
and improves sleep, activity, and cognition. Because this is a behavioral intervention, it is difficult to blind the 
subjects to the active vs sham treatment, and the biofeedback coach who provides the training must know the 
condition to which the subject is assigned. However, all others in the study are blinded to the training. The 
specific aims are: 1.) Determine whether HRV-B increases HRV coherence and reduces pain or stress relative 
to a sham treatment among Veterans with chronic, nonmalignant, axial neuromusculoskeletal pain; 2.) 
Determine as an exploratory aim whether HRV-B reduces insomnia, fatigue or depressive symptoms, and 
increases sleep and activity and improves cognition among patents relative to sham treatment. We 
hypothesize that HRV-B will (1) reduce self-reported pain and stress ratings, (2) improve objective measures of 
actigraphic sleep parameters (sleep latency, duration, efficiency, fragmentation), rest/activity rhythms 
(dichotomy index, interdaily stability) and cognitive function (reaction time, attention); and (3) alleviate self-
reported fatigue and depression symptoms. Patients from both groups will be randomized to our previously 
established HRV-B or sham protocol (n=40 each), and will complete a baseline assessment, 6 weekly training 
sessions, a post-training assessment, a 4-week booster training and assessment, and an 8-week post-training 
follow-up evaluation, for a total of 10 visits to the research site. Portable, hand-held, data-logging devices will 
be used to practice attaining HRV coherence at home by the active treatment group, while those in the sham 
training group will get a ‘stress squeeze ball’. Standard methods will quantify HRV coherence and other HRV 
measures, and validated instruments will be used to assess pain and stress. Wrist actigraphy is a valid, 
quantitative, and noninvasive procedure that will be used to characterize insomnia via continuous recordings 
collected 24-hrs/day over three 1-week periods (at the baseline, post-treatment, and 8-week follow-up 
assessment). A small battery of tests measuring attention and reaction time will assess changes in cognitive 
performance. Data analyses will apply linear models for repeated measures to evaluate the effect of HRV-B on 
study outcomes, and on treatment persistence, after adjusting for confounding factors. For each measure we 
will calculate three estimates of change for each patient (post-training minus pre-training, 4-week booster 
minus post-training, and 8-week follow-up minus the 4-week booster assessment). Path analyses will examine 
direct and indirect relationships between HRV-B, the primary and exploratory outcomes, and personal or social 
factors that may modify these relationships. This study will be the first to examine HRV-B for pain management 
among Veteran chronic pain patients. This study is directly responsive to a national VHA/DoD Task Force’s 
recommendations to provide complementary, integrative therapies for pain management to Veterans. 
 
 

 
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1.0  BACKGROUND AND SIGNIFICANCE 
 
Pain is the most common reason for seeking health care in the United States, impacting ~116 million 
Americans and generating >$600 billion in medical care costs.1  Chronic pain is a significant issue among 
Veteran patients and is one of the most common reasons for seeking care among Veterans of all wars.2,3   
Among all Veterans, musculoskeletal pain is the most commonly diagnosed medical problem.4  Pain patients 
experience improved quality of life when pain is controlled and they are more likely to successfully manage 
other aspects of their daily lives.5  Because the prevalence and intensity of pain increases with disease 
progression, and since pain impacts multiple domains of quality of life (QoL),6,7 the ability to manage pain 
becomes increasingly important over time for medical patients of all types.   
Pain initiates a stress response that increases sympathetic output leading to autonomic imbalance and 
psychological stress; physical and psychological stress in turn exacerbates pain, thus creating a self-
perpetuating cycle of autonomic dysregulation that facilitates persistent pain.8  Stress from chronic pain leads 
to loss of the glucocorticoid (GC)-mediated negative feedback control of the hypothalamic-pituitary-adrenal 
(HPA) axis, producing positive HPA drive and down‐regulation of central and peripheral GC receptors. In 
addition, peripheral cytokine pro-inflammatory signals from nociceptive receptor chronic stimulation is sent to 
the  spinal dorsal horn and ascends to brainstem, and gated in thalamus prior to cognitive appraisal in primary 
somatosensory cortex.  This creates pain awareness.  As a result, monoaminergic (dopamine, norepinephrine 
and epinephrine) neurons in the brainstem that normally descend to the spinal cord and ‘brake’ nociceptive 
transmission become depleted during chronic pain.9  This loss of inhibitory monoaminergic tone to the spinal 
cord leads to enhancement of pain (‘central sensitization’). In what is now a feed-forward system, the loss of 
GC inhibition of pro‐inflammatory cytokines leads to proliferation of peripheral inflammatory agents, which are 
nociceptive transmitters, further contributing to central pain sensitization and an HPA driven stress response.10  
At the same time, down‐regulation of GC negative-feedback modulation from chronic stress also promotes 
depressed mood.11  Thus, chronic pain can induce both stress and depression (Figure 1, ‘Disease Pathway’). 
Pain Medication & Addiction. Pain remains a major healthcare problem despite advances in treatment of major 
medical illnesses and illness-related pharmacological pain management.12  Opioid analgesics such as 
morphine and hydrocodone and its derivatives effectively produce analgesia through antagonism of the 
receptors of naturally occurring opioids that are released from the periaqueductal gray region of the central 
nervous system in response to pain. However, the use of opioid analgesic medication poses an increased risk 
of physical and psychological addiction, as well as a number of other side effects including intolerance, 
hyperalgesia, hormonal imbalance, respiratory depression, constipation, nausea, vomiting, drowsiness, and 
itching13  Nonetheless, Vicodin is the most prescribed medication in the United States.14  Overdose deaths 
from opioid painkillers have more than quadrupled since the turn of the century, approaching 20,000 annually, 
and opioid overdose is the leading cause of accidental death in several states.15  Many Veterans with mental 
health diagnoses receive prescription opioids for pain-related conditions, which can compromise their mental 
health.16  An effective non-pharmacological pain therapy that reduces the usage of opioid medications would 
be a significant benefit to chronic pain patients.   
Non-Pharmacological Pain Treatment.  Many veterans in chronic pain use avoidance strategies to dampen the 
intensity of pain in their lives. This avoidance is most clearly seen in behaviors and movement. Painful 
activities are avoided in an attempt to decrease the overall experience of pain. Though behavioral avoidance 
can lessen pain in the short term, it can also cause long term problems and decrease QoL.17 Complementary 
therapies, such as Heart Rate Variability (HRV) Biofeedback (HRV-B), that can help improve both physical and 
psychological well-being have been advocated for integration with conventional therapies because many 
medical patients are not satisfied with mainstream treatments in relieving their pain symptoms and the 
attendant erosion of their QoL.18   
Our proposal is designed to test whether HRV-B is feasible and effective in increasing HRV coherence and 
reducing perceived pain, stress, depression, and insomnia among Veterans suffering from chronic physical 
pain. Our path analyses will examine both direct and indirect relationships between HRV-B, the primary and 
exploratory outcomes, and other individual or social factors that may modify this relationship. The relationship 

 
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between autonomic dysfunction and disrupted sleep has not been thoroughly examined. Our path analyses are 
thus important from a practical standpoint because HRV coherence and sleep represent potentially modifiable 
risk factors that can be targeted to reduce symptom burdens and improve QoL for Veteran patients. In addition, 
we will implement the use of portable, hand held finger plethysmographs (personal stress relievers) to reinforce 
the HRV-B training and promote practice outside the clinic. We have obtained encouraging preliminary results 
by incorporating this technique in our other research. In our path analysis, we will identify factors predicting 
persistent HRV-B effects at the 8-week post-treatment follow-up. This information will be useful in designing 
future interventions. The use of HRV-B to normalize autonomic function represents a novel, complementary 
strategy that engages patients in their own care and is directly responsive to the VHA/DoD Pain Management 
Task Force’s recommendations to adopt complementary pain management strategies (including HRV-B) to 
alleviate pain among Veterans.19  This application addresses several CSR&D priority research areas (Pain 
Management, Complementary and Alternative Medicine, Patient-Centered Care) and coincides with a 
nationwide VHA/DoD initiative to include complementary, integrative, therapies into pain management for 
Veterans.  To our knowledge, the use of HRV-B to improve chronic pain symptoms in a Veteran population has 
not previously been studied in a randomized clinical trial.     
Chronic Pain, Stress, Depression and Autonomic Imbalance.  HRV is a valid, noninvasive measure of 
autonomic function with both physiological and psychological attributes.  Reduced HRV is an established risk 
factor for mortality due to cardiovascular or other chronic diseases.20,21 For example, about 80% of advanced 
cancer patients exhibit autonomic 
dysregulation,20,22 and several recent studies 
report increased mortality risk among patients 
with reduced HRV.20-26 Furthermore, emotional 
disruption caused by a stressful experience is 
characterized by a chaotic, disordered pattern 
of inter-beat intervals as well as an increased 
heart rate, which lowers HRV (Figure 2, top 
panel). Irregular heart beat rhythm due to 
disordered inter-beat intervals, reflects  
sympathetic nervous system over-activity and 
low HRV coherence. In contrast, emotional 
balance and a feeling of well-being results in 
an ordered, sine wave-like pattern of inter-beat 
interval changes and a narrow peak in the low 
frequency (LF) range of the power spectrum, 
centered around 0.1 Hz, which equals a period 
of 10 seconds, or 6 cycles per minute (Figure 
2, bottom panel).27  
 
As a measure of the interplay between the excitatory sympathetic and the inhibitory parasympathetic nervous 
systems, HRV has a clear relationship with pain sensitivity and perception. Linkages between HRV alterations 
and various types of pain have been documented. Appelhans and Luecken found an inverse relationship 
between HRV and sensitivity to cold thermal pain measured as ‘unpleasantness’, and ‘threshold for barely 
noticeable pain’. 28 In patients with fibromyalgia, HRV was a predictor of pain perception, pain anxiety, and 
physical health functioning.29 The pattern of HRV in patients under general anesthesia changed to reflect 
decreased vagal tone when a surgical stimulation was painful, even though the patient was not conscious.30 
Alternatively, a reduction in visceral pain during esophageal balloon distension was associated with higher 
HRV.31  
The autonomic nervous system exhibits a distinct circadian rhythm as do several cardiac measures including 
heart rate and blood pressure.13 The heart rate increases in the morning at or after waking, reaches peak 
between 10 am to 12 pm, and then gradually begins to decline later in the day, maintaining a low level during 
night. During sleep, increases in parasympathetic tone and HRV predominate. Despite these associations, few 

 
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studies have examined relationships between disrupted sleep and autonomic function.16,32 In a study of 13 
healthy male volunteers randomized to 5 nights of partial sleep deprivation (<5 h), or 5 nights of control sleep 
(>7 h), those with sleep deprivation had a statistically significant increase in sympathetic activity as indicated 
by an increase in low frequency (LF) and decrease in high frequency (HF) components of HRV.33 The results 
were consistent with another study that reported increased sympathetic and decreased parasympathetic HRV 
measures after 36-hours of total sleep deprivation.32  
Depression is relatively common in patients with coronary heart disease (CHD) and its association with 
autonomic nervous system dysfunction has been investigated among CHD patients. HRV is lower among 
depressed patients with either stable CHD, or among those with a recent acute coronary event, compared with 
non-depressed patients.34 Studies examining HRV and depression have expanded into other patient 
populations. Another recent meta-analysis concluded that depression in the absence of cardiovascular disease 
is also associated with reduced HRV, and that HRV decreases with increasing depression severity.35 Reduced 
HRV is an established predictor of increased mortality risk both in cardiovascular or other chronic disease 
patients, and in the general population.20,21  
 
Loss of HRV coherence is also associated with 
PTSD.  Our recently published meta-analysis of 
HRV and PTSD found that HRV variables are 
associated with PTSD, and these associations can 
be interpreted in terms of a basic and widespread 
model of PTSD as a disorder comprising hyper-
arousal of the sympathetic nervous system (see 
Preliminary Studies).36  
 
HRV Biofeedback.  Improvements in HRV can be 
therapeutic. Complementary therapies, such as 
HRV-B that can help improve both physical and 
psychological well-being have been advocated for 
integration with conventional therapies. HRV-B is 
an interactive procedure whereby patients increase 
HRV in real time by practicing focused attention, 
positive emotion, and resonant frequency 
breathing. Previous research suggests that HRV-B interventions may be useful for reducing symptoms of: 
chronic pain,28,31,37,38 anxiety,39-41 depression,37,42 post-traumatic stress disorder (PTSD),43,44 heart disease,45 
and insomnia.46   
HRV-B is a complementary, non-pharmacological technique for improvement of HRV and autonomic function. 
It is an interactive procedure whereby patients monitor their HRV patterns in real time while practicing focused 
attention, positive emotion, and resonant frequency breathing. The resonant frequency of breathing (RFB) is 
the respiratory rate producing the highest HRV in the LF band; RFB is necessary for attainment of HRV 
coherence.47 HRV coherence is achieved when beat-to-beat intervals increase and decrease in a smooth 
rhythm, producing a spike around a 10 second period (0.1 Hz on the fast Fourier transformation to the power 
spectrum) of the instantaneous heart rate (Figure 2). It is defined according to consensus guidelines as the 
ratio of power in the LF peak to the remainder of power in the HRV spectrum.27,44,48  Using HRV-B, patients 
learn how to produce HRV patterns indicative of higher vagal parasympathetic tone. With practice, patients 
acquire the ability to enhance their HRV coherence on demand (see Figure 2). High coherence has been 
associated with a favorable psychological state, including improved affect, cognition, and executive function.27   
HRV-B had analgesic effects on experimental heat pain threshold and tolerance scores in normal subjects.49 
The HRV-B technique has been used in several clinical settings to improve HRV and reduce pain symptoms of 
fibromyalgia,37 back pain,38 and chronic stress-related neck pain.41 In our own work50 with chronic pain patients 
from our DVAMC Pain Clinic, several pain and stress perception ratings were lowered by HRV-B compared to 

 
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a control group of treatment as usual. Thus, the neurobiology underlying analgesia induced by this method of 
complementary, non-pharmacological, psychophysiological self-regulation appears to be different from other 
commonly used techniques involving distraction. The potential benefits of improving autonomic function among 
medical patients has been acknowledged.20,21,26,51      
Chronic pain is a significant issue among Veterans, and to our knowledge, this will be the only study other than 
our pilot study50 to evaluate HRV-B for the amelioration of pain in this population. The alleviation of persistent 
pain, fatigue, insomnia or depression via improved HRV coherence may provide a direct clinical benefit to 
Veteran pain patients. Our study will examine both direct and indirect relationships between HRV-B, the 
primary and exploratory outcomes, and other individual or social factors that may modify this relationship. We 
will also develop an integrated statistical model of autonomic dysfunction as a potential underlying 
psychophysiological process that can be targeted to improve the management of pain. This will include the 
identification of predictors of disrupted sleep and rest/activity rhythms. If our aims are achieved, our study has 
importance from a practical standpoint because HRV coherence and sleep represent potentially modifiable risk 
factors that can be targeted to reduce symptom burdens and improve QoL for pain patients. The integrated 
path analysis model derived from this study will aid in the development of more effective intervention strategies 
for addressing symptoms and thus targets for the design of future projects. If this study’s hypotheses are 
supported, we will seek funding for a full-scale intervention to comprehensively examine whether HRV-B 
therapy alleviates pain in a wider population of Veterans with pain.   
1.1.  Preliminary Studies 
We are now conducting a randomized, single blind, sham-controlled study funded by the Department of 
Defense (DoD) to examine the effectiveness of HRV-B in alleviating PTSD symptoms. Three groups of combat 
veterans are being studied: (1) PTSD patients who received active HRV-B training  (n=30); (2) PTSD patients 
who received sham HRV-B training (n=30); and (3) Veterans without PTSD who were assessed only at 
baseline and received no HRV-B training. Training lasted six weeks.  Participants were assessed Pre-training, 
immediately Post-training, and 8 weeks after training. To date, 73 participants have been enrolled, with 32 
assigned to the Sham group, 29 to the active Biofeedback group, and 12 to the Control group.  Data analysis is 
ongoing and results are being prepared for submission for review for publication. 
Effect of HRV-B on HRV Coherence.  In this ongoing study, the time domain variable RMSSD was used as an 
indicator of coherence because it 
correlates very highly with vagal tone and 
parasympathetic output (r=0.853, 
p<0.001).52,53 Linear mixed model analysis 
of RMSSD for Group (Sham vs. HRVB)  x 
Time Period of Assessment (Pre-, Post-, 
Follow-up) showed that the interaction 
effect was significant, F(5, 125)=2.50, 
p=.034. Follow-up testing of the interaction 
effect revealed that the Group main effect 
was statistically significant, HRV-B > 
Sham, 60.2 (43.6) vs. 46.1 (37.3), 
F(1,127)=4.66, p=.033.  Contrasts within 
the HRV-B group revealed that Pre-
training RMSSD was statistically lower 
than combined Post-training and Follow-
up, 44.1 (22.8) vs. 72.6 (51.4), t(57)=-2.58, 
p=.013, but Post-training was not 
statistically different from Follow-up, 74.8 (63.2) vs. 69.7 (32.8), t(57)=0.346, p=.730. In summary, active HRV-
B increased HRV coherence and the increase persisted until the Follow-up period (Figure 3). 

 
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Effects of HRVB on Veterans with Chronic Pain. This study50 determined the effectiveness of HRV-B as a pain 
management intervention for DVAMC Veterans with chronic pain by comparing pre- and post-intervention pain 
and stress scores among two 
groups of randomly assigned 
Veterans that met the chronic 
pain inclusion criterion. One 
group received HRV-B plus 
treatment as usual and the 
other group received only 
treatment as usual. The 
outcomes of this study were 
HRV coherence and four scale 
scores (pain, emotional 
distress, activity limitation, and 
stress) that were obtained from 
two previously validated 
psychometric instruments (BPI 
and PSS, respectively).  
Veterans receiving HRV-B 
training were coached to use 
constructive processes to 
engage and manage internal 
sensations of pain rather than rely on strategies of avoidance. All subjects received a baseline and a follow-up 
assessment after 6 weeks of HRV-B training or treatment as usual. Retention for this pilot study was 100%.  
The HRV-B group received five weekly HRV-B training sessions. Treatment effects were analyzed with 
ANCOVA of follow-up scores by group using baseline scores as covariates.  Eight Veterans completed the 
HRV-B intervention and six Veterans in the usual treatment group returned for a follow-up assessment.  The 
mean baseline values for the two groups were not statistically different for any of the measures.  In the HRV-B 
group, there was a statistically significant increase in  HRV coherence and a reduction among each of the four 
outcome measures for pain or stress post-HRV-B training relative to the group that only received usual 
treatment (all p <0.05,  Figure 4).  Results from this study indicate that HRV-B can effectively reduce the 
negative perceptions of pain.  Data obtained from this investigation were used to develop statistical  power and 
sample size estimates since they are most directly relevant to the proposed intervention. 
Effect of HRV-B on PTSD among Combat Veterans. In 2010, we 
examined the effects of HRV-B on combat Veterans with PTSD 
in a small pilot study at DVAMC (n=10).  In our published 
findings, we reported that HRV-B significantly improved HRV 
coherence as well as measures of attention and immediate 
memory (ATTN/IM).44 In our current DoD-funded research, 
baseline PTSD symptoms were not significantly different 
between the active and sham training groups (Table 1). Linear 
mixed model analysis54 of PTSD rating (CAPS) for the Training 
Group by Time interaction was statistically significant, F(5, 
127)=2.60, p=.028; furthermore the Pre- to Follow-up difference within the HRV-B group was significant. Paired 
t-test within the HRV-B group of Pre- to Follow-up was statistically significant, mean=16.7, t(12)=4.66, p=0.001 
(ηp2=0.663).  These treatment effect was well beyond the accepted reliable change score of 12 points 
reduction in CAPS55; a reduction of 15 points on the CAPS is considered clinically beneficial.56  These results 
indicate that PTSD severity was reduced by HRV-B training compared to the Sham group, and the benefit 
persisted for 8 weeks after training ended. 
 
Table 1.  Effect of HRVB on PTSD (CAPS) 
Training 
CAPS 
Pre- 
Post- 
8-week 
Follow-up 
Sham  
Mean 
76.1 
69.9 
72.9 
SE 
n 
4.3 
32 
5.2 
23 
7.1 
18 
HRV-B 
Mean  
77.5 
53.1 
60.8 
SE 
n 
3.7 
29 
7.7 
16 
7.7 
12 

 
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Effect of HRV-B on Depression in Combat Veterans with PTSD. Depression (BDI-II) was also measured in our 
current DoD-funded research.  The repeated measures contrast of Group x Time for the effect of HRV-B on 
Depression for Pre- to Post-Training was statistically significant, (F (1,28)=6.29, p=0.02 (ηp2=0.173)).  The 
reduction in BDI-II in the HRV-B group compared to Pre-training was statistically significant, (mean=5.9, 
t(16)=1.82, p(1-tail)=.045 (ηp2=0.180)), while the BDI-II change in the Sham training group was not, p(1-
tail)=.23. These results indicate that depression was reduced by the HRV-B treatment compared to the Sham 
training at the Post-Training assessment.  A change of 5 points in the BDI is considered to be a reliable and 
clinically significant change.57 
Effect of HRV-B on Sleep in Combat Veterans with PTSD. To evaluate the effect of active versus sham HRV-B 
on sleep in our on-going DoD-funded PTSD study, we examined the self-rating of sleep difficulty from the PCL-
M. This rating is on a scale of 1 to 5, where 1 means no 
difficulty and 5 corresponds to extreme sleep difficulty. 
Participant’s scores on sleep difficulty, analyzed using a 
linear mixed model, indicated a statistically significant 
Group by Time interaction  (F(5, 127)=2.41, p=.028) with 
better post-training sleep ratings in the HRV-B group 
(Figure  5).  Thus, there is preliminary evidence from our 
study among PTSD patients to suggest that active HRV-
B training reduces self-reported sleep difficulties. Data 
from more sources measuring sleep quantity and quality 
is required to fully evaluate this possibility. This study of 
HRV-B effectiveness for PTSD is still in progress and no 
conclusions can be drawn at this time. However, the 
results are encouraging and suggest that HRV-B may 
improve the negative impact of PTSD on sleep among 
combat Veterans.    
HRV & Shift Work among DVAMC Nurses.  Few studies have examined autonomic dysregulation or the 
circadian characteristics of HRV in relation to shift work. Dr. Burch  recently completeda study of the 
relationship between shift work and HRV among DVAMC 
nurses. Participants were studied over one 36-hour period 
coinciding with the last two shifts of their work week. The night 
nurse’s schedule consisted of non-rotating 12-hour shifts from 
7:00 PM to 7:00 AM for 3 or 4 consecutive days in a 2-week 
period. Day nurses worked a similar schedule from 7:00 AM to 
7:00 PM. Each participant wore a portable, data-logging HRV 
monitor (Firstbeat Bodyguard, Jyväskylä, Finland) starting at 
the beginning of one 12-hour work shift and stopping at the end 
of the final shift of a given work week. This light weight heart 
rate monitor attaches directly to the skin with two chest 
electrodes and continuously logs the R-R heart beat interval in 
milliseconds. Data were processed using KUBIOS software. Hourly averages of frequency- (HRV coherence, 
VLF, LF, HF power, peak frequency, peak frequency power) and time-domain variables (heart rate mean and 
standard deviation, NN50, pNN50, RMSSD) were compared among two groups of nurses working either 
permanent day (n=7) or night (n=11) shifts using repeated measures ANOVA. Results indicate that nurses 
working permanent day shifts had a clear circadian pattern of HRV coherence coinciding with their sleep period 
(hours ~15-23), whereas those working permanent night shifts had no apparent overnight amplitude in the 
circadian rhythm of their HRV coherence (Figure 6). These results suggest a pattern of autonomic 
dysregulation among nurses working permanent night shifts.  
Sleep Actigraphy. The value of wrist actigraphy for sleep characterization is recognized in both clinical and 
research settings as an alternative to polysomnography (PSG), which is the most definitive method but can be 

 
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more expensive, labor-intensive, and burdensome to participants. We have studied actigraphic sleep profiles 
among: shiftworkers, patients with sleep apnea or cancer, and in the general population.58-60,74 We studied 
shiftwork tolerance and melatonin metabolite excretion among manufacturing workers on permanent day, 
evening or night shifts, and used wrist actigraphy to document greater sleep disruption among night workers 
relative to day workers.58 Recently, we compared the accuracy of sleep measures obtained via wrist- or hip-
mounted actigraphs with PSG recordings. We also attempted to improve the correspondence between 
actigraphic and PSG sleep using a novel sleep scoring algorithm (spline regression). Compared to original 
wrist actigraphy data, spline-modified wrist actigraphy had better agreement with PSG-defined sleep. Thus, 
spline regression has the potential to improve sleep estimates obtained via wrist actigraphy.61  
 
1.2.  Specific Aims 
 
This study fulfills the national VHA/DoD Task Force recommendation that complementary, integrative therapies 
for pain management be provided to Veterans. Chronic pain elicits stress which increases sympathetic output 
fostering autonomic nervous system imbalance, an overextended stress response, fatigue, depression and 
insomnia. Heart Rate Variability (HRV) is a measure of the interplay between the sympathetic and 
parasympathetic nervous systems, and thus is a useful and easily-measured index of autonomic balance that 
has a relationship to chronic pain effects. HRV biofeedback (HRV-B) is a novel, biobehavioral procedure that 
restores normal autonomic balance. Through HRV-B, patients increase parasympathetic cardiac output and 
restore autonomic balance via induction of  ‘HRV coherence’.  Our pilot study indicated that HRV-B produced 
coherence and alleviated self-reported ratings of chronic pain and stress among Veterans attending our Pain 
Clinic.50  The proposed clinical intervention will further test hypotheses that HRV-B increases HRV coherence, 
reduces self-reported pain, stress, depression, fatigue, and insomnia, and improves cognition among Veterans 
with chronic pain.  
 
The specific aims are to: (1) conduct a randomized, sham-controlled, pilot intervention trial to determine 
whether HRV-B increases HRV coherence among chronic pain patients (n=40 each for the HRV-B and sham 
treatment groups; total N=80 patients); (2) determine whether HRV-B reduces self-reported pain and stress 
among chronic pain patients. The primary endpoints include HRV coherence, pain (Brief Pain Inventory or 
BPI), and stress (Perceived Stress Scale or PSS).   
 
Furthermore, chronic stress is associated with disrupted circadian rest/activity rhythms and domains of quality 
of life (QoL) including fatigue, insomnia, and reduced physical and social functioning.62,63 Interventions that 
relieve pain thus represent a novel therapeutic target for normalizing dysfunctional rest/activity rhythms and 
these QoL domains among pain patients. We are also interested in assessing the effects of HRV-B on 
cognitive function in pain patients. Thus, our secondary exploratory objectives are to determine if HRV-B: 
(1) improves sleep and rest/activity rhythms; (2) alleviates self-reported fatigue and depression; and (3) 
improves cognitive function (reaction time, attention). Circadian endpoints will be measured as actigraphic 
parameters of sleep (e.g., duration, efficiency), and rest/activity (e.g., dichotomy index, interdaily stability), and 
with the Insomnia Symptom Questionnaire (ISQ); fatigue will be assessed using the Multi-Dimensional Fatigue 
Inventory (MFI), which assesses general physical and mental fatigue and motivation. Depression will be 
assessed via the Beck Depression Inventory II (BDI-II). Cognitive function will be measured with a cognitive 
battery comprised of the Paced Auditory Serial Addition Test (PASAT), the Rey Auditory Verbal Learning Test 
(RAVLT), and the Psychomotor Vigilance Test (PVT).    
 
2.0  STUDY DESIGN AND METHODS 
 
Overview. The proposed intervention will use a previously approved, standardized HRV-B training protocol.44  
The primary (HRV coherence, pain, stress) and exploratory outcomes (insomnia, fatigue, depression, 
cognition) will be assessed at baseline, after the 6 weekly treatments, at the 4-week post-training booster, and 
at the 8-week post-training follow-up. Standardized procedures will characterize HRV coherence and other 
frequency- or time-domain HRV measures, and validated instruments will be used to assess pain64,65 and 

 
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stress.66 Sham intervention subjects will have pulse and respiration monitored but not receive active training; 
instead they will view a static, relaxing nature picture on a computer screen. We will provide portable data-
logging devices for practicing HRV-B at home, and sham devices. Wrist actigraphy will characterize insomnia 
via continuous, 24-hour/day personal monitoring of rest/activity rhythms at 1-week intervals coinciding with the 
baseline, post-training, and 8-week follow-up assessments only.58,67-69  
 
Data analyses will test for differences in primary and secondary endpoints at baseline, after 6 weeks of active 
versus sham HRV-B treatment, at the 4-week post-training booster/assessment, and 8 weeks after training. 
Analyses will be based on intent to treat with two-sided tests. The effect of HRV-B on coherence and other 
HRV variables will be analyzed using linear mixed models for repeated measures data from sequential time 
assessments, and a between-subjects factor to evaluate the intervention after adjusting for potential 
confounding factors (e.g., age, standard therapy, medications, co-morbid disease). Baseline relationships 
between HRV coherence and endpoints will be examined using multiple regression models. Few studies have 
examined the multivariate relationship between pain, autonomic dysfunction, stress, depression, sleep, 
rest/activity rhythms, fatigue, and cognition. Path analyses will be used to model interrelationships between 
HRV-B, primary and exploratory outcomes, and behavioral or social factors that facilitate the persistence of 
HRV coherence and pain management effectiveness. 
 
2.1  Study Population and Recruitment 
 
Subjects from the DVAMC Pain Clinic who are: English literate, ≥18 years old, of any race, ethnicity, or sex 
meeting inclusion and exclusion criteria will be recruited. An additional inclusion criterion is that the patient 
reports having chronic pain as defined by our screening questions. Our initial recruitment effort will target 
patients in the Dorn VAMC Pain Clinic. The Pain Clinic runs an active Outpatient Interdisciplinary Pain 
Program, which consists of weekly sessions of Nursing Education, Nurse Triage, Behavioral Medicine (ACT, or 
Acceptance and Commitment Therapy), Physical Therapy, Pharmacy Education, Biofeedback, and 
Acupuncture. This patient population is appropriate for the proposed HRV-B intervention. If our accrual 
milestones are lagging recruitment goals, we will expand our search for eligible patients to include the 
Rehabilitative Medicine, Rheumatology, or Primary Care departments at the DVAMC. Our approved poster 
(Section 2.1) will be used to advertise the study at the DVAMC, the Columbia Veterans Center, the University 
of South Carolina Veterans Association office, or similar Veteran venues. Permission from local offices will be 
obtained prior to posting the advertisement.   
 
Inclusion criteria for the Pain Clinic includes patients with: chronic, non-malignant, axial neuromusculoskeletal 
pain (e.g., osteoarthritis, degenerative discs or joints, scoliosis, spinal stenosis, spondylolisthesis, compression 
fractures, or other musculoskeletal conditions with primarily nociceptive pain, ICD-9: 715.x, 721.x, 722.x, 723.x, 
724.x, 729.x, 733.x, 738.x, 756.x). A Recruitment Coordinator will be responsible for screening and applying 
inclusion and exclusion criteria to potential research subjects. 
 
We will exclude patients who are: not appropriate for the intervention from a safety standpoint; have a 
condition that can bias HRV measures; or those not able to cognitively complete the protocol. We will exclude 
patients with: a) history of arrhythmias requiring medication and/or hospitalization, including supraventricular 
tachycardia or atrial arrhythmias (e.g., atrial fibrillation); b) patients with a pacemaker or automatic implantable 
cardioverter-defibrillator; c) history of an acute coronary syndrome, revascularization, thrombolytic or other 
therapy related to ischemic heart disease; d) uncontrolled hypertension (systolic blood pressure <140 mmHg, 
diastolic blood pressure <90 mmHg), although patients with well-controlled hypertension (no change in 
medications for 6 months) will not be excluded; e) history of heart transplant or cardiovascular surgery within 1 
year; f) patients receiving beta-adrenergic antagonists; g) patients receiving non-dihydropyridine calcium 
channel blockers; h) patients receiving an antagonist of the renin-angiotensin-aldosterone system are eligible if 
their medication profile is stable; i) patients with New York Heart Association class 3 or 4 congestive heart 
failure; j) history of seizure disorder or use of antiseizure or anticonvulsant medication; k) cognitive impairment 
(e.g., dementia), or a history of acquired neurocognitive deficit, or central nervous system or neurological 

 
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disorder (e.g., Gulf War Syndrome); l) moderate or severe head injury or stroke; m) evidence of active 
substance abuse or dependence (alcohol or tobacco use will not be an exclusion, patients will be asked to 
report their use via questionnaire); n) life history of bipolar, psychotic, panic or obsessive-compulsive disorder 
(history of depression will not be an exclusion); o) previous experience with HRV biofeedback. Eighty 
participants will be recruited into the study. All participants will be Veterans being treated at the DVAMC. 
 
Recruitment will follow a multi-step process of: 1.) obtaining a HIPPA wavier to identify active patients who are 
eligible for the study based on inclusion and exclusion criteria; 2.) informing potential participants of the nature 
of the study by letter, and excluding those who decline from the potential participant list; 3.) for those who do 
not decline, making a follow-up telephone call to ascertain interest in study, and if the patient expresses 
interest in the study; 4.) screening them during the same telephone call for chronic pain and any other 
inclusions and exclusions not ascertained from the medical record; and 5.) obtaining informed consent from 
patients eligible and willing to participate. The PIs will obtain a HIPPA wavier to access the docket of patients 
with a new or current pain diagnosis (within 6 months). This list will be narrowed to eligible patients based on 
medical record review. A staff recruitment coordinator will be responsible for patient screening and recruitment.  
 
Patient contact information will be obtained for this refined list of patients, and each patient will receive a 
packet of information about the study that includes a cover letter from the DVAMC Pain Clinic Director co-
signed by the study PIs, and a study brochure. The letter and brochure will briefly describe: the nature of this 
study in lay terms, the extent of participation, confidentiality requirements, and contact information for the 
recruitment specialist and PIs. The letter will be written at no more than a sixth grade reading level using plain 
language. The letter will contain a coded, pre-addressed, postage-paid envelope that provides a ‘no contact’ 
response. The letter will also state that if a negative response is not received by the patient within two weeks, 
then a recruitment specialist will contact the individual by telephone to ascertain their willingness to participate.  
Patients returning postcards who decline further contact will be removed from the eligible contact list.  As an 
alternative, potentially eligible patients will be referred to a study coordinator by the nursing staff attending 
patients in the Pain Clinic. A study brochure has been developed to provide information about the study to 
patients in the Pain Clinic. It may be provided to potentially eligible patients by medical or research staff. The 
study brochure was developed based on information in the consent form, and it is presented in Appendix VI. A 
poster that will be used to advertise the study  is presented below.  

 
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The study investigators will meet with staff in each 
clinic to provide information on the study protocol, 
design, and eligibility requirements. The recruitment 
procedure for referred patients will be the same as 
that described above. Eligibility will be checked, 
patients will then receive the packets and will be 
telephoned to ascertain interest in the study and 
perform the chronic pain eligibility screening (Pain 
Screening Questionnaire, Vanderbilt University 
Medical Center, Center for Quality Aging, Nashville, 
TN). The pain screening instrument assesses pain 
using these questions: 1.) Do you have pain 
anywhere right now? 2.) Does pain ever keep you 
from sleeping at night? 3.) Does your pain ever keep 
you from participating in activities/doing things you 
enjoy? 4.) Do you have pain every day? Chronic pain 
is present if the patient answers yes to questions 1-3, 
or to question 4 alone. During the study, patients will 
be asked to self-report the duration of their chronic 
pain. In addition, the patient’s medical record will be 
examined (via the CPRS) to determine the duration of 
time between their first pain clinic appointment and 
enrollment in the study. These variables will be 
included as covariates in the data analysis. Once 
enrolled in the study, we will record the pain 
treatments received by participants from the medical 
record for use in data analysis.    
 
2.2  Intervention and Data Collection 
 
An appointment for the baseline assessment will be made among patients with chronic pain who meet other 
eligibility criteria and express an interest in participation; scheduled at a mutually agreed upon date and time.  
Study staff will meet participants at the DVAMC in order to obtain informed consent, conduct an interview to 
include baseline ascertainment of primary outcomes, and initiate baseline actigraphy.  Participants will receive 
a $30 reimbursement for each of the sessions they attend. A total of 10 sessions is planned: baseline (pre-
training), six weekly training sessions, post-training evaluation, a ‘booster session’ 4 weeks after post-training, 
and an 8-week post-treatment follow-up session (4 weeks after the booster session) for a total of 10 sessions. 
(= $300 for protocol completion, Figure 7). Patients attending the DVAMC Pain Clinic will be recruited over ~36 
months of the 48 month period funding for randomization to an active HRV-B (n=40) or sham (n=40) protocol. 
Given the purpose of the study (pain and stress reduction) and the accessibility of chronic non-malignant 
neuromusculoskeletal pain patients, we anticipate a recruitment rate of ~75%. Research personnel will 
annotate each participant’s CPRS medical record that the Veteran has enrolled in this research. Participants 
will be requested not to change their ongoing pain treatment for the duration of the study unless it is urgently 
indicated. The Veteran’s pain clinical provider will be advised to forego changes in the Veteran’s pain treatment 

 
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plan during the Veteran’s participation in the study unless a change in the pain treatment plan is imperative. 
Determination of the need to change the pain treatment plan will be made by the Pain Clinic Director (Dr. Katz) 
or his designee. When a change in pain treatment is made, the participant will remain in the study and 
specifics of the change in treatment will be recorded for later evaluation in the statistical analysis. Non-
participants will be asked to provide the following 
information: age, race/ethnicity, education, sex, 
and pain rating. This information will be compared 
among participants and non-participants to 
assess potential selection bias. Reasons for 
nonparticipation or noncompliance will be 
summarized and we will evaluate whether this 
information may suggest selection bias and 
protocol modifications in future research. The 
Rehabilitative Medicine and Rheumatology 
departments at the DVAMC will serve as 
alternative recruitment sites, if needed, to expand 
the pool of eligible patients. The study brochure 
and poster may be used to provide information 
about the study to patients attending these 
clinics. A project schedule is presented in Table 
2. A subject sign-in sheet for tracking site visits is 
presented below. A list of frequently asked questions with answers is presented in Appendix VI. 
 
 
Figure 7. Intervention Timeline 
Booster 

 
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Baseline HRV Assessment. Resting physiological measures to be recorded for 20 minutes at baseline. We will 
use our autonomic testing system (budgeted start-up equipment) to precisely determine HRV at the four time 
periods (pre-testing, post-testing, booster, 8-week follow-up). Testing will be performed in a standardized, 
recognized, and quantified manner that is non-invasive. By monitoring the amplitude changes of the peak 
frequency in a power spectrum of the inter-beat intervals in synchrony with respiration, it is possible to detect 
when a specific respiration rate is causing an increase in the peak frequency output, which defines the 
Table 2.  Project Schedule 
Project Month 
1-6 
6-9 
10-36 37-42 
42-48 
Obtain Institutional Review Board and Research & Development 
Committee approvals 
X 
 
 
 
 
Hire Research Associate (Project Coordinator) and Graduate 
Research Assistant 
X 
 
 
 
 
Finalize clinical protocol for HRV-B and sham treatments, subject 
recruitment and retention, actigraphic data collection, and database 
development, validation and quality control 
X 
 
 
 
 
Purchase equipment and supplies for data collection and storage 
X 
 
 
 
 
Data management and quality control review 
X 
 
 
 
 
Initiate recruitment, data collection, and database development 
 
X 
 
 
 
Continue and complete subject enrollment 
 
X 
X 
X 
 
Complete database; Data management and quality control review 
 
 
 
X 
 
Perform statistical data analyses 
 
 
 
X 
 
Data management and quality control review 
 
 
 
 
X 
Prepare and submit manuscripts for publication 
 
 
 
 
X 

 
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resonant frequency of breathing.  Resonance frequency breathing produces the highest HRV in the LF band. 
Cardiovagal (parasympathetic) function is monitored and HRV is derived using heart rate response to deep 
breathing and transformation of tachygram recordings, procedures that the investigator (JPG) is experienced 
with, using easy-to-perform, and time efficient measurements. Heart rate will be recorded via dry electrode 
wrist straps and respiration will be monitored via a Piezo-respiratory transducer. Software integrated into the 
HRV physiological monitoring system will run on a laptop computer and be used to de-artifact raw data and 
calculate the FFT power spectrum of HRV for each patient (Appendix  I). This will provide data for frequency- 
(HRV coherence, VLF, LF, HF power, peak frequency, peak frequency power) and time-domain HRV 
measures (heart rate mean and standard deviation, NN50, pNN50, RMSSD).  HRV coherence is defined in 
accordance with consensus guidelines.27,44,47,48  It is estimated by calculating the ratio of power in the LF peak 
to the remainder of power in the spectrum.  
Random Assignment to the 6-week HRV-B treatment or sham intervention group will occur following the 
baseline assessment. We will use a permuted block randomization procedure, with a block size of 4 and no 
stratification. Thus, for example, assuming 20 patients are to be assigned either to Sham (S) or Biofeedback, 
there would be 20 blocks of 4 patients, and two patients will be assigned each to the (S) treatment and two to 
the biofeedback by randomly selecting one of six possible permutations of the two treatments in blocks of four. 
The order of the blocks will be randomly generated. The treatment assignment will be determined before any 
patient is enrolled into the study and the documentation and assignment of patients will be kept confidential 
and known only to the PI (JPG). Only the Biofeedback professional, who delivers the treatment, will know the 
treatment status of the participant; other study personnel will be blinded to treatment status. 
 
HRV-B Training. HRV-B training will follow our previously established, standardized protocol that is consistent 
with recommendations of the Biofeedback Certification Institute of America (BCIA), consisting of a 15-minute 
resting period that includes HRV recording, and a 25-minute biofeedback training and coaching or sham 
period. The resting periods are the same for both the HRV-B treatment and sham groups. Participants will be 
affixed with HRV monitoring equipment and will be instructed to sit quietly for 15 minutes in a relaxed posture, 
viewing a static nature scene on a computer monitor, without any instruction, coaching or biofeedback. 
Coaching or instruction will start after the 15-minute resting period. Patients in the sham condition will be asked 
to ‘just relax’ or ‘take it easy’ for the remainder of the 25-minute session. Patients in the HRV-B group will be 
coached on how to achieve HRV coherence. 
 
HRV-B training is conducted in an interactive manner using a computerized system to monitor and display the 
individuals’ HRV patterns in real time while their heart rate, measured as blood volume pulse (BVP), is 
recorded with a fingertip photoplethysmograph (PPG). Visual HRV feedback (either a quantitative display or 
animated challenge games) is provided as participants practice attention focusing, resonant frequency 
breathing, imagery, and the induction of positive emotion. The resulting changes in HRV patterns are readily 
observed by the subject. The visual feedback enables associations to be formed between these techniques 
and HRV patterns indicative of higher vagal parasympathetic output. Before HRV-B training, the biofeedback 
trainer discusses individual issues and the training goals of the participant.  The physiological connection 
between resonant frequency breathing and heart rate is introduced. This is reinforced with corrective breathing 
instruction and help finding the resonant frequency (~6 respirations/min). Dual monitors also display the HRV 
power spectrum in real-time so that the participant can visualize the impact  of proper breathing on the 
coherence peak a 0.10 Hz. Positive emotion and interactive visual images are then introduced. This interactive 
process allows the participant to customize a sustainable system, with occasional shifts back to the power 
spectrum screen to observe the 0.10 Hz HRV peak.  During the recording period after coaching, subjects view 
a static nature scene on the computer while their heart rate is measured with the plethysmograph and recorded 
(Appendix III). 
 
Home Practice. The biofeedback coach will instruct participants in both groups to practice their training at 
home a minimum of 10 minutes a day, and participants will be given a log to record their daily home practice. 
Home practice self-report data will be incorporated into the analysis to examine the potential dose-response 

 
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effects of home practice. Information will be solicited in the questionnaire regarding the factors that contributed 
to or detracted from home practice for inclusion in the analysis. One goal of this intervention is to 
spontaneously and consistently achieve HRV coherence on demand outside a clinical environment. Therefore, 
participants will be provided with a portable colorimetric finger PPG (emWave® hand held personal stress 
reliever; dimensions similar to a cell phone; weight: 2.2 ounces) for home practice and use between weekly 
HRV-B training sessions. Home HRV-B practice period data from the device can be compared to self-report 
and further analyzed with regard to the outcomes (see Appendix III, IV).    
 
Intervention Integrity. Several procedures will be used to evaluate treatment integrity. The intervention will 
follow the HRV-B protocol adopted by the BCIA, and BCIA certification for HRV-B or its equivalent training will 
be required for trainers in this study. Protocol compliance integrity will be monitored and supervised by 
research staff and Dr. Burch , and via communication between them and the certified biofeedback professional 
(Jane Areve). We will ask patients about whether they thought they were enrolled in the active or control 
condition. We will then summarize these data along with the number and duration of participant visits over 
time, and compare these values between the sham and treatment groups. Contrasts between the sham and 
treatment condition for outcomes measured at baseline, post-treatment, booster, and at the 8-week follow-up 
will allow us to evaluate the accuracy and delivery of the intervention. Measures of HRV coherence will allow 
for direct, quantitative assessment of participant performance and receipt of intervention. 
 
Sham Treatment. To control for the laboratory environment or other placebo effects, participants assigned to 
the sham HRV-B training group will be scheduled by the biofeedback professional for a weekly clinic meeting 
during the 6-week period of the project that matches the timing and duration of the active HRV-B sessions. The 
sham procedure has been used successfully in our ongoing study of HRV-B among PTSD patients for the past 
three years. During these lab visits, sham training participants will have their heart rate, respiration, blood 
pressure and blood pressure volume recorded during a 15-minute resting period, but no active training or 
coaching will be provided.  
 
During the ~25 minute coaching period, the Biofeedback professional has the participant sit quietly in front of 
the computer screen with the fingertip PPG in place, but the recording input is switched off so no information 
on heart rate is delivered to the recording equipment. No active biofeedback coaching is delivered regarding 
resonant frequency breathing, imagery, or positive emotion induction. The Biofeedback professional uses 
passive relaxation statements such as: ‘try to relax’ and ‘please sit quietly’ during the coaching period. During 
the passive 15-minute HRV recording period after the coaching session, the recording input of the PPG is 
switched on, and the subjects view the same static relaxing nature scene on the monitor as is presented to the 
HRV-B group. For home  practice, participants in the sham group are given a ‘squeeze stress ball’ and asked 
to use it throughout the normal activities of the day. Subjects in the sham group will be asked to self-report the 
frequency and duration of squeeze ball use.     
 
Primary & Exploratory Questionnaire Outcomes. In addition to HRV coherence, the primary outcomes (pain, 
stress), as well as the related exploratory measure, will be assessed at baseline, after the 6-week intervention 
period, during the booster session and at the follow-up session. Validated instruments will be used to 
administer a structured questionnaire focusing on: 1). pain (BPI64,65) and stress (PSS66); 2). the exploratory 
outcomes (sleep, fatigue, depressive symptoms, cognitive performance); 3). well-being (coping, social 
support); 4). lifestyle (chronotype, social jetlag); 5). socio-demographic and personal characteristics (age, 
height, weight, education, income, physical activity; 6). occupation (current or previous job title, combat history, 
shift work); 7). supplemental use of pain medications including over the counter anti-inflammatory agents [e.g., 
NSAIDs], as well as alcohol and tobacco consumption; and 8). whether they have a pending worker 
compensation claim or personal injury litigation related to their pain or other symptoms. Some items from the 
medical record (e.g., age, body weight) will be compared with questionnaire data to assess reporting validity. 
Questions focusing on the duration of symptoms (e.g., sleep disruption, fatigue) will be integrated into the 
study instrument (e.g., the duration of: sadness, fatigue, stress, or sleep disruption). Co-morbid diagnoses of: 
hypertension, diabetes, cardiovascular disease, stroke, obstructive sleep apnea or other sleep disorders, or 

 
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post-traumatic stress disorder (PTSD), and use of prescription medications (hypnotics and other sleep aids, 
antidepressants, anxiolytics), and will be assessed by chart review after obtaining a HIPAA release of medical 
information as part of the informed consent process. Questionnaires are presented in Appendix V.    
 
Pain. The BPI was developed by the World Health Organization (WHO) specifically for use among cancer 
patients, and it has since been widely adopted for assessment of clinical pain and pain treatment effectiveness 
in a variety of clinical and research settings.64,70 Among the barriers that result in under treatment of cancer 
pain, one key consideration is the adequate pain characterization. The BPI is an assessment tool pain that 
measures both pain intensity (sensory dimension) and the interference of pain in the patient's life (reactive 
dimension). The BPI has demonstrated reliability and validity across different cultures and languages, and it 
has been applied to studies of clinical pain assessment, as well as epidemiological investigations and research 
on the effectiveness of pain treatment.64,70  
 
Pain Catastrophizing Scale (PCS). The PCS was developed to facilitate research on the mechanisms by 
which catastrophizing impacts the pain experience. Items on the PCS were drawn from previous experimental 
and clinical research on catastrophic thinking in relation to pain experience (http://sullivan-
painresearch.mcgill.ca/).71 The factor structure has been replicated in several investigations. The PCS total 
score is computed by summing responses to all 13 items. PCS total scores range from 0-52. The PCS requires 
a reading level of approximately Grade 6 and can be completed and scored in less than 5 minutes. It is a 13-
item instrument derived from definitions of catastrophizing described in the literature. The PCS instructions ask 
participants to reflect on past painful experiences, and to indicate the degree to which they experienced each 
of 13 thoughts or feelings when experiencing pain, on 5-point scales with the end points (0) not at all and (4) all 
the time. The PCS yields a total score and three subscale scores assessing rumination, magnification and 
helplessness. The PCS has been shown to have adequate to excellent internal consistency (coefficient alphas: 
total PCS = .87, rumination = .87, magnification = .66, and helplessness = .78).71  
Stress. The PSS is the most widely used psychological instrument for measuring the perception of stress. The 
original version, published in 1983,72,73 has been refined over the years into its current form and is available a 
public domain instrument. It measures the degree to which situations in one’s life are appraised as stressful. 
Items were designed to tap how unpredictable, uncontrollable, and overloaded respondents find their lives. The 
items are easy to understand and general in nature, and hence relatively free of content specific to any 
subpopulation group. PSS scores are correlated with self-reported health, health behaviors, smoking status, 
and help seeking  behavior.73 The PSS exhibits excellent construct validity and reliability.66  
 
Sleep & Rest/Activity Rhythms.  Wrist actigraphy is a non-invasive, quantitative, and validated method that will 
provide a primary means by which circadian disruption is assessed in this study. Weekly actigraphic monitoring 
will be performed once each at baseline, post-treatment, and at the 8-weeek follow-up using a standardized 
protocol.14,74-76 Actiwatch-Spectrum monitors (with built-in ambient multiple spectrum light sensor, event 
marker, digital watch, and off-wrist detection capability, Respironics, Bend, OR) will be used in this study. They 
are similar in size, weight, and appearance to a wristwatch. Monitors will be worn on the non-dominant wrist, 
recording activity and light exposures at 1-minute intervals. Subjects will wear the monitors 24-hours per day 
for three 1-week intervals (baseline, post-treatment, and at the 8-week post-training follow-up). Participants will 
be asked to follow their usual sleep-wake schedules and maintain a log of when they go to bed and awaken on 
days when they wear the actiwatch. At the end of each 1-week actigraphic monitoring period, a study 
technician will retrieve the monitor from the subject during their weekly training session. Alternatively, 
participants can mail the actiwatch to research staff using a pre-labeled, postage-paid envelope that will be 
provided to the participants, or a study investigator can retrieve the monitor from the individual’s residence. 
These methods have all been successfully implemented in previous studies among DVAMC patients and 
elsewhere.14,77  Actigraphic sleep parameters have been previously validated,67,68,78,79 and 1-week intervals 
provide for ample sleep or rest/activity rhythm assessment.79 A weekly average of sleep metrics (latency, 
duration, efficiency, fragmentation, wake time after sleep onset [WASO], daytime nap duration) will be 
quantified using the manufacturer’s accompanying software (Appendix  II).  

 
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Sleep disruption will be ascertained using the Pittsburgh Sleep Quality Index (PSQI), which is a self-
administered 19 question instrument, categorized into 7 components, that assesses sleep duration as well as 
latency, frequency, and severity of sleep problems with a possible range of 0-21. The 7 components are 
subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of 
sleep medication, and daytime dysfunction. The Cronbach’s alpha coefficient for all component scores was 
0.83 which indicates high internal consistency. Those with disorders of initiating and maintaining sleep and with 
disorders of excessive somnolence had statistically significantly higher scores compared to control subjects, 
which indicates good construct validity. A cut-point score of 5.0 indicated a sensitivity of 90% and a specificity 
of 87% in identifying those with a sleep disorder.80 One advantage of the PSQI over other instruments is that it 
provides information on overall subjective sleep quality and includes several items related to snoring and the 
induction of sleep apnea or hypopneas, which can influence heart rate, a primary study outcome. We will 
modify the time scale for reporting on sleep quality to the previous week to coincide with planned wrist 
actigraphy measures. We will augment the use of this instrument by performing CPRS queries for each 
participant (with a HIPAA release) to identify diagnostic codes for insomnia or other sleep disorders.     
 
Using actigraphy data, insomnia may be defined using the following cut-off values: sleep onset latency >12 
minutes, total sleep time <440 minutes, WASO >25 minutes, sleep efficiency <92%, or the number of 5-minute 
wake episodes ≥2.69 The phase and amplitude of circadian rest/activity rhythms will be characterized via 
previously described non-parametric (interdaily stability, intradaily variability, relative amplitude, 24-hour 
autocorrelation coefficient) and cosinor statistics (acrophase, mesor, amplitude, amplitude/mesor ratio, 
goodness of fit).67,68,81 Ambient light exposures collected via actiwatches will be summarized using the same 
descriptive, cosinor, and nonparametric statistics.  
 
Ambulatory Heart Rate Variability Monitoring. In a limited number of participants, a 
convenience sample (n=15-30 total) will be asked to wear a data-logging ambulatory 
heart rate monitor (Firstbeat Bodyguard 2; Firstbeat Technologies, Jyväskylä, 
Finland) for two 1-week periods that coincide with data collection for the wrist activity 
monitor (i.e., at baseline and during the post-training week). The heart monitor is 
lightweight, portable and noninvasive (see photo). It attaches to the skin with a snap 
electrode and does not require a belt around the body. There is no need for the 
participant to operate or adjust the monitor in any way, they only need to wear it. Note: it should be removed 
when taking a shower, or while bathing or swimming (extra skin patch electrodes will be provided to re-attach 
the device). Participants will wear this device 24 hours per day. The data record allows for time- (mean and std 
HR, NN50, pNN50, and RMSSD) and frequency-domain variables (VLF, LF and HF power, peak frequency, 
peak frequency power, and coherence) to be generated for comparison with actigraphic sleep data and other 
exploratory research. Research staff will hand out and collect bot the heart rates and wrist actigraphy monitors 
at the same time for each participant. Data are downloaded for processing with a cable attached to the 
computer’s USB port, and the battery is recharged in a similar manner. Participants will be provided with 
written and verbal instructions on appropriate use (no showering, bathing or swimming) and with extra leads for 
re-attaching the monitor after showering, etc.     
 
Depression & Fatigue will be ascertained using several valid instruments. Fatigue will be assessed using the 
Multi-Dimensional Fatigue Inventory (MFI), comprised of 20 questions that assess general, physical, and 
mental fatigue, and reduced motivation and activity. It also has good internal consistency (Cronbach alpha: 
>0.75) and high convergent validity confirmed via strong correlations with several other validated 
instruments.82,83  Depressive symptoms will be ascertained using the Beck Depression Inventory II (BDI-II).84-86  
The BDI-II is a 21-item self-report instrument that asses the existence and severity of symptoms of depression 
as listed in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 
Fourth Edition (DSM-IV).  The BDI-II has excellent clinical sensitivity (Cronbach alpha: 0.92).84 Its construct 
validity has been established, and it readily differentiates depressed from non-depressed patients.87  
 

 
Version 12, 15 May 2020, with Amendment 12  
 
Cognitive Performance.  A short and simple battery consisting of three validated neuropsychological tests will 
be administered at the 4 test periods: the Paced Auditory Serial Addition Test (PASAT), the Hopkins Verbal 
Learning Test (HVLT), and the Psychomotor Vigilance Test (PVT).  These PASAT and HVLT have alternate 
forms and the PVT has no significant practice effect. This battery of assessment instruments has sufficient 
reliability to detect changes over repeated test sessions. Brief descriptions are provided below. 
 
Paced Auditory Serial Addition Test (PASAT). The PASAT is a measure of cognitive function that assesses 
auditory information processing speed and flexibility, as well as calculation ability. It was developed in 1977 
and is now very widely used in clinical and research applications to assess cognitive function. In the PASAT, 
single digits are presented every 3 seconds and the patient must add each new digit to the one immediately 
prior to it. Several alternate forms have been developed that are publically available to minimize familiarity with 
the stimulus items when the PASAT is repeated on more than one occasion.  Administration time is 
approximately 5 minutes. The score for the PASAT is the total number correct out of 30 possible answers.  The 
PASAT has demonstrated high split-half reliability and evidence for convergent and divergent validity with good 
sensitivity for deficits in the areas of auditory information processing speed and flexibility.  
 
Hopkins Verbal Learning Test. The HVLT is a measure of verbal learning and memory, in which the participant 
is presented with a list of 12 semantically categorized words and is asked to recall as many as he/she can. 
This process is repeated twice more, and the score is equal to the total number of words recalled across all 
three trials. The test takes approximately 5-10 minutes to administer. There are several alternate versions to 
eliminate practice effects across repeat test sessions. The norms are in the public domain and available 
without cost.  
 
Psychomotor Vigilance Task (PVT). The psychomotor vigilance task (PVT) is a widely used and well-validated 
test of sustained-attention and reaction-time that measures the speed with which subjects respond to a visual 
stimulus.  The PVT correlates with lowered alertness, slower problem-solving, decreased psycho-motor skills, 
and increased rate of false responding. The PVT is popular because of its ease of administration and scoring, 
simple metrics, and convergent validity.  The PVT is a simple task where the subject presses a button as soon 
as a light appears on the computer screen. The light will turn on randomly every few seconds for 5 minutes. 
The main measurement of this task is to assess reaction time and how many times the button is not pressed 
when the light is on. The purpose of the PVT is to measure sustained attention, and give a numerical measure 
of level of arousal. The program is publically available at no cost. 
 
Lifestyle and sociodemographic characteristics will be assessed using instruments developed in our previous 
studies among Veterans.44,77,88  Chronotype refers to the self-reported characterization of individuals with 
inherent morning or evening preference (or neither).89,90 This trait has been associated with intrinsic circadian 
period,91 and the circadian timing of numerous psychophysiological processes,92-95 including sleep,96,97 arousal 
and alertness,98 mood,99 and the secretion of catecholamines,100 and cortisol.101 The Munich Chronotype 
Questionnaire (MCTQ)102 will be used to ascertain self-reported chronotype and sleep/wake timing on free and 
work days. Disparities between chronotype and social schedules can result in chronically desynchronized 
sleep-wake timing or ‘Social Jetlag’,103,104 which will be quantified as the absolute difference between mid-sleep 
on weekdays and weekends, after adjusting for weekend make-up sleep.104 Other potential modifiers of the 
HRV-B treatment that will be assessed include: social support,105 and coping.106  
 
Saliva Specimens. Saliva samples (~1/2 tablespoon) will be collected by each participant; once at baseline, 
once at the post-treatment assessment, and another at the final follow-up visit. One ~2 mL saliva sample will 
be collected each time using the simple, noninvasive passive drool method with Oragene® DNA collection kits 
(model: OGR-500, DNA Genotek, Ontario, Canada, http://www.dnagenotek.com).107 The vial has a fill line for 
precise volume collection, and salivary DNA is immediately stabilized by a preserving reagent. Samples will be 
frozen at -80oC for long term archive. DNA can be purified from the archived specimens using a standard 
ethanol precipitation method from commercially available kits. The median yield for a 2 mL sample using this 
device is 110 µg DNA. Good quality DNA will be defined as a 260/280 UV absorbance ratio of 1.7-2.0. These 

 
Version 12, 15 May 2020, with Amendment 12  
 
specimens will serve as a valuable resource for future research targeting biological processes that may 
mediate of modify the treatment effect. Another ~2 mL native saliva sample (without added reagent) will be 
collected for recovery of proteins or peptides using the same collection procedure.    
 
The samples will be archived in frozen storage for future research. Samples will be coded with a unique 
number that will not include participant’s name or any other personal information. Only the Principal 
Investigators, or a study Co-Investigator, will have access to the code that links the sample to their identity. 
The samples will be stored indefinitely in a -80C freezer for future research purposes at the DVAMC Building 
9A, basement. Analyses that are under consideration include the study of genes involved in pain or other study 
endpoints, or responses to the HRV-B treatment. This may include candidate gene or genomic sequences, 
gene expression, or genetic modifications (e.g., epigenetic changes, sequencing). Analyses may also include 
immune system markers, hormones, or other compounds or microbes (e.g., bacteria) in saliva. Some sample 
analyses will be performed in the laboratory of Dr. Angela Murphy, 6439 Garners Ferry Rd., School of 
Medicine Building 1, Room C27, Columbia SC 29209. Sample analyses also will be performed by EpigenDx, 
96 South St, Hopkinton, MA 01748. These measures will be for exploratory research and not for clinical use. 
Therefore, individual results will not be provided to participants. Use of these samples for future research is 
noted on the consent document. 
 
2.3  Data Analysis Plan  
 
Statistical Power & Sample Size. Preliminary data for HRV coherence and the pain (BPI), and stress (PSS) 
scales from our HRV-B pilot study among DVAMC chronic pain patients were used to estimate statistical 
power for the proposed primary study outcomes (HRV coherence, pain rating, negative emotion, activity 
limitation, perceived stress scale). Two-sided, two sample t-tests were used for the statistical power 
calculations. The analysis for the proposed study will use more sophisticated methods that tend to reduce 
variance in the dependent variables and thus increase power, thus what is presented here is likely 
conservative. An overall significance level of 0.05 was used with correction for multiple testing via the 
Bonferroni method for the 5 outcomes (adjusted significance level = 0.01; 0.05/5).  Our pilot data indicate that 
at baseline, no statistical differences were observed between treatment groups for any outcome. The 
detectable effect differences between treatment and sham groups with corresponding standard deviations are 
presented in Table 3. We assumed equal variances in the differences between pre- and post-treatment 
comparisons for both groups. A sample size of 25 in each group will attain a statistical power of at least 84.5% 
to detect the expected effect differences between treatment groups, and groups sizes of 40 patients will 
provide statistical power exceeding 95% for comparisons between treatment groups for each outcome. Thus, a 
sample size of 40 patients per group would achieve ample statistical power even with retentions on of 55-60%. 
 
Table 3. Estimated Power for Differences Between HRV-B Treatment & Sham Interventions 
Sample 
Size per 
Group 
Negative Emotion 
(4.5±10; 
15.5±10)* 
Activity limitation 
(4± 9.5; 14± 9.5)* 
Pain Rating 
(1.8±5.0; 
10±5.0)* 
Perceived Stress 
(-1.2±4.5; 
4±4.5)* 
HRV 
Coherence 
(-0.03±0.15; 
-0.20±0.15)* 
22 
82.3% 
78.1% 
99.6% 
86.5% 
84.9% 
25 
88.1% 
84.5% 
99.9% 
91.4% 
90.2% 
30 
94.1% 
91.7% 
99.9% 
96.2% 
95.4% 
40 
98.7% 
97.9% 
99.9% 
99.3% 
99.1% 
*Average pre-post change ± SD in the control group; and average pre-post change ± SD in the treatment group, respectively 
 
Data Quality Control. Data collected by physiological sensors will be encoded and recorded without any 
identifying information. Analysis and interpretation of data files also will be conducted without reference to any 
identifying information. Questionnaire data will be labeled using the participant’s identification number and will 
not have personal identifying information anywhere on the form. Once collated, coded questionnaire data will 

 
Version 12, 15 May 2020, with Amendment 12  
 
be entered into a password protected computer for further processing and analysis. We will use the double key 
entry method of data verification (or equivalent) to help ensure data entry accuracy. 
 
Data Security. Multiple steps will be taken to ensure complete confidentiality of the study data, and 
confidentiality will be maintained to the fullest extent allowed by law. Data files will be stripped of personal 
identifiers and replaced with coded participant identification numbers that will be used to link data files from 
multiple sources (e.g., questionnaires). Once collated, coded data will be entered into encrypted VA 
workstation computers for further analysis and processing. Data files will be stored on an access-controlled 
data server (network access to the server is by password for a limited set of study personnel: Building 106, 
Room E111; \\v07.med.va.gov\cms\vhacms\vhacmsburchj for Dr. Burch.. To minimize risk of loss of 
confidentiality, only authorized personnel will have access to research data. Participants will be informed that 
the local IRB may review de-identified or identified study data. The key linking participant identifiers to the 
coded data will be maintained by the Contact PI in a locked file cabinet at the DVAMC. Hard copy records with 
patient identifiers (e.g., signed consent forms) will be maintained in the Contact PI’s office at the DVAMC in a 
locked file cabinet, accessible only to authorized study personnel (Building 106, Hard copies with patient 
identifiers will be stored in Room E111A). Consultants will not have access to any identifiable data. Data 
analyses will be performed at the DVAMC using coded data with personal identifiers removed, using coded 
data via an approved data use agreement with the Department of Epidemiology and Biostatistics, Arnold 
School of Public Health, University of South Carolina, or using coded data without identifiers via a secure VA 
Virtual Private Network (VPN) workspace (see below). All study personnel will maintain current VA training 
requirements (e.g., data security, human subjects protection). If an investigator or research staff member 
terminates their work on the project, their access to the data will become restricted, and the Information 
Security Officer will be notified. Data will be maintained in a secure manner until it is no longer needed or until 
its maintenance is no longer legally required. Data destruction will be performed by deleting electronic files and 
by shredding paper files.  
 
VINCI Workspace. The VA Informatics and Computing Infrastructure (VINCI) is a Department of Veterans 
Affairs (VA) Health Services Research & Development (HSR&D) resource center that provides a secure, 
central analytic platform for performing research and supporting clinical operations activities. It is a partnership 
between the VA Office of Information Technology (OI&T) and the Veterans Health Administration Office of 
Research and Development (VHA ORD). VINCI includes a cluster of servers for securely hosting suites of 
databases integrated from select national VA data sources, as well as statistical software packages for data 
analysis. VINCI servers for data, applications and virtual sessions are physically located at the VA Austin 
Information Technology Center (AITC) in Austin, Texas. This secure enclave with 105 high-performance 
servers and 1.5 petabytes of high-speed data storage has multiple layers of security and disaster recovery to 
prevent data loss. To ensure the protection of Veteran data, VINCI maintains compliance with the guidelines 
set forth by Veterans Health Administration (VHA) Handbook 1200.12, Use of Data and Data Repositories in 
VHA Research, and other applicable VA and VHA policies and regulations. In addition, VINCI has undergone 
security certification activities in support of obtaining an Authorization to Operate (ATO). Access to VINCI 
resources are approved in accordance with the requirements of National Data Systems (NDS), VHA Handbook 
1200.12, Use of Data and Data Repositories in VHA Research, and all other applicable VA and VHA policies 
and regulations. All data transferred from VINCI is subject to audit for compliance. 
 
VA-credentialed investigators are granted access to study-specific data along with tools for analysis and 
reporting in the secure, virtual working environment through a certified VHA network computer within the VA.  If 
not working within a VA or VHA hosted office environment containing VA network access, researchers may 
apply for and then access VINCI through an approved VPN and Remote Desktop application. The remote 
computing environment enables data analysis to be performed directly on VINCI servers, offering a number of 
advantages: uniform security standards for access; a common point of entry for all investigators who use the 
data; tools for analysis and reporting; tighter and more consistent control of data quality; and the ability to 
standardize and update terminology and format as technology and methodology improve. Software used for 

 
Version 12, 15 May 2020, with Amendment 12  
 
data analysis in the VINCI workspace, whether provided by VINCI or developed by the study team, will run in 
remote virtual desktop sessions on VINCI servers within the Austin Information Technology Center. 
 
For this study, coded project specific data without personal identifiers may be uploaded to VINCI servers by 
secure file transfer protocol as directed by VINCI data managers. Study data maintained on VINCI servers will 
be kept in accordance with the Department of Veterans Affairs Record Control Schedule 10-1 (RCS 10-1). 
Data transferred to VINCI servers will be coded using a participant ID number as described above (see Data 
Security section) so that none of the data will be personally identifiable. Upon completion of the research 
project, the study Principal Investigators in conjunction with the VA Information Security Officer (ISO), and in 
accordance with VA policy, will ensure that, study data will be returned to the DVAMC. The study Principal 
Investigators have the responsibility for security of study data. Only explicitly authorized study personnel will 
have access to project data. When study personnel are no longer part of the research team, the study Principal 
Investigators will amend the data access request to terminate that person’s access to all study data and notify 
the VA Information Security Officer of such action. VINCI data managers and VA OI&T personnel not under the 
purview of the study Principal Investigators control the servers, network, processors, firewall and software in 
the VINCI environment, including access rights granted to study personnel. Study specific data stored on 
VINCI servers will be located at the Austin Information Technology Center, 1615 Woodward St., Austin, TX 
78772-0001. The specific server where the data are stored within the VINCI environment will be chosen by 
VINCI personnel. The server name and location within the Austin Information Technology Center may be 
changed at any time at the discretion of VINCI personnel. 
 
Statistical Analyses. Analysis and interpretation of data files will be conducted without reference to 
any personal identifying information. We will begin all analyses with an inspection of all variables for 
completeness, any missing values will be tabulated and range checks will be used to identify observations that 
need to be validated. Once the data set has been finalized, a locked version will be created to use for all 
analyses. Descriptive statistics will be applied to questionnaire scores, their potential predictors, demographic 
and lifestyle variables to evaluate their characteristics and assess their general inter-relationships (e.g., via 
correlations for numeric measures, or by frequencies among treatment groups for categorical variables). These 
analyses will provide an understanding of relationships among variables and will aid in planning subsequent 
analyses. The main analyses pertaining to aims 1 and 2 will consist of linear mixed models for repeated 
measures data to evaluate the treatment effect on outcomes of interest. We will also implement secondary 
exploratory path analyses to evaluate direct and indirect effects of the treatment on the primary (HRV 
coherence, pain, stress), and secondary (insomnia, fatigue, depression, cognition) outcomes.   
For each of the outcomes, there will be three measurements per subject (post-training minus pre-training, 4-
week booster minus post-training, and 8-week follow-up minus the 4-week booster assessment). To test the 
effect of HRV-B relative to controls, we will apply linear mixed models for repeated measures data. Models will 
include the group (intervention or sham), time, and the interaction between group and time as the main 
independent variables. The effect of the intervention on the outcome could be non-linear in time, thus we will 
evaluate the inclusion of time as a nominal or ordinal variable and choose the specification that has the best fit 
in terms of the model assumptions and the BIC statistic. Covariates to be considered in the models include (but 
are not limited to: age, type and duration of usual pain treatment or medication, or other outcomes that may 
modify the effect (e.g., stress, pain, sleep). We will use a variable selection procedure to screen and identify all 
variables that differ among the treatment and control groups (initial selection set at p<0.15), and we will include 
them in the final statistical model if their inclusion alters the effect estimate for HRV-B by ≥10%.  Linear mixed 
effect models will be implemented using the PROC MIXED procedure in SAS (v9.3, SAS Institute). The 
conceptual model of each linear mixed model is:  
, 
where Y is the dependent variable, X is the independent variable of interest (or a vector of covariates), si is a 
subject-specific random effect, and t represents the outcome assessment time points (baseline, follow-up). The 
β coefficients measure each independent variable’s effect. If time is included as an ordinal variable, 
0
1
2
3
ij
ij
j
ij
j
i
ij
Y
X
t
X
t
s
β
β
β
β
ε
=
+
+
+
×
+
+

 
Version 12, 15 May 2020, with Amendment 12  
 
significance between treatment and control groups will be tested using Wald tests for appropriate β 
coefficients. If time is included as an nominal variable, we will use least squared means to test for significant 
differences between treatment and control groups at each of the post-intervention time points (post-training, 
booster, 8-week follow-up). In this situation we will use a Bonferroni adjusted alpha level = 0.005 (0.05/10), 
which results in at least 78% power for all of the outcomes with 25 per group to account for attrition (data not 
shown). 
 
We will use "intention to treat" (ITT) analyses on all subjects without regard to level of compliance. ITT analysis 
is based on the assumption that once a subject is randomized to a group, data for that subject will enter into 
the final analysis. ITT assumes unrelated treatment events are equally likely across groups and that outcome 
data are complete. All data from non-adherent and dropout subjects are included; therefore, ITT provides 
conservative effect estimates. ITT is most appropriate for randomized, controlled clinical trials involving lengthy 
treatment protocols when an effective treatment arrests progression of disease. Because recommendations for 
treatment of primary and secondary outcomes will likely continue to include care (i.e., controls still receive 
usual care), ITT analysis will better reflect what will be observed if HRV-B is introduced into clinical practice. 
This analysis answers the question: What is the effect of HRV-B on outcome measures in a situation where 
individuals range from fully compliant to non-compliant? In this pilot intervention, however, we also are 
interested in the actual effect of HRV-B on the outcomes. In ancillary analyses, we will perform multiple 
imputations for subjects with missing data (see below). These post hoc analyses will answer the question: 
What is the effect of full HRV-B training on the outcomes? This will provide a more accurate picture of HRV-B 
effects because it focuses only on subjects who completed the training. 
 
Missing Data. Inevitably, missing data will arise and a variety of approaches will be considered to address this 
issue. First, our integrated team will meet and discuss the hypothesized nature of the missingness 
mechanism.108 For example, if the subjects have data that are missing completely at random or missing at 
random (MAR) then multiple imputation (MI) procedures can be used (e.g., “PROC MI”).109  When missingness 
is suspected to be non-ignorable, then a model for the missingness mechanism must be employed.110,111 In all 
models, sensitivity analyses will be performed to test the robustness of the estimates to the missing data 
assumption (if any). We will create a number of locked datasets (e.g., 10) with imputed values that will be used 
in all analyses that require MI. This will ensure that all MI analyses are based on the same imputed data. 
 
Path analysis is a useful statistical tool for modeling how multifaceted processes combine to influence specific 
outcomes. We will apply path analysis to evaluate the direct and indirect effects of HRV-B on the pain-related 
study outcomes (pain, stress, fatigue, insomnia, depression) via changes in HRV coherence (Figure 8). This 
analysis will estimate the direct effect of the intervention on the post-training minus pre-training measurements 
of the study outcomes. There are three paths 
that are depicted by the arrows in Figure 8. 
The path model will determine the path 
coefficients, variance explained, and statistical 
significance of direct and indirect (modifier) 
effects. Modifiers may include: race, income, 
lifestyle, duration or quality of hand held stress 
reliever use, social jetlag, family or work 
history, physical activity, major life events, 
coping strategies, or social support. Other 
paths that consider the influence of modifiers 
or confounders will be explored to determine if they improve model fit. PROC CALIS in SAS will be used; 
satisfactory model parameters will be identified using a Bentler comparative fit index >0.93. This integrated 
model with appropriate modifiers and mediators will identify variables that contribute to, or detract from, the 
treatment effect on HRV coherence and pain, stress, fatigue, depression and insomnia. The above SEM is a 
relatively simple structure with only 6 baseline parameters per outcome, thus with a sample of 60 (30 per 

 
Version 12, 15 May 2020, with Amendment 12  
 
group) the study will have the required minimum number of observations based on the 10:1 rule-of-thumb for 
sample size with SEMs.112 
 
2.4.  Data and Safety Monitoring Plan 
 
The PIs and research staff will conduct ongoing monitoring the progress of this pilot intervention trial and the 
participant safety. This Data and Safety Monitoring Plan will be used to assess on-going participant safety. No 
invasive procedure will be used in the study. The participants will be fully informed of the procedures, including 
notification that their medical records from the DVAMC will be examined. Participants will voluntarily sign an 
Informed Consent and HIPAA Authorization approved by the DVAMC IRB before participation begins. 
 
Data will be collected from the participants by face-to-face interview, having them complete objective 
psychometric instruments such as questionnaires and validated tests, and via the widely used procedure of 
HRV Biofeedback. In addition, data will be collected by record review of medical and service history obtainable 
from the Dorn VAMC Electronic Medical Record (CPRS), after authorization has been signed by the patient. 
Data collection will be specifically for the proposed research. Data collection time points for HRV and 
questionnaire will be at pre-training (T-1), post-training (T-2), 4-week post-training booster (T-3), and 8-week 
post-training follow-up (T-4). Baseline measurements will occur after the patient has been screened for 
eligibility, and consented. Randomization will occur after baseline assessment. All variables will be measured 
at each time point except demographics (T-1 only), and saliva (T-1, T-2, T-4). Measurements will be the same 
for both the active and sham intervention groups. 
 
Potential Benefits. This study is designed to elicit relief from pain and related symptoms, although the study it 
is an experimental intervention and subjects may not obtain direct personal benefit. However, benefit to future 
patients is possible. Patients will receive an acknowledgement of their time and effort in participating in the 
study and they will receive financial compensation for each assessment and training session according to the 
schedule of incentives. The knowledge generated by this study will provide an understanding of the potential 
benefit of HRV-B on pain, stress, fatigue, sleep and quality of life for Veterans with chronic pain, and may 
inform clinical practice and programmatic standards concerning appropriate ways to assist with pain 
management, sleep disturbance and fatigue among DVAMC patients. 
 
Potential Risks. No significant physical, social, or legal risk is anticipated with this study. All proposed 
procedures have been widely used over a long period of time by the investigators and/or others. HRV-B is a 
noninvasive procedure. There have been a few cases of relaxation-induced anxiety noted (i.e., with 
progressive muscle relaxation), but these effects are not associated with HRV-B or controlled breathing. Other 
negative reactions that have been associated with other modalities of biofeedback-assisted relaxation (such as 
sensation of heaviness, feelings of sadness, disturbing thoughts, or mind wandering) are rare, mild, and brief. 
All these reactions can be prevented by an experienced biofeedback professional with precautions, patient 
education, and careful monitoring.  
 
Risks to confidentiality are minimal since all data will be coded with a study ID. Study ID and patient list will be 
kept separate, and the file linking study IDs with personal identifiers will be maintained in a locked file cabinet 
of password protected electronic file in one or both of the PI’s offices. Data safety and monitoring is described 
below. 
 
No adverse events were observed during  other HRV-B studies. . Subjects will be informed that the 
experimental protocol will be provided in addition to their treatment as usual, will not affect their usual care, and 
no other intervention is available as an alternative intervention at the DVAMC. Minimal risks are anticipated for 
the subjects. 
 
Protection Against Risks. Veterans seeking care at the DVAMC who meet the eligibility criteria will be recruited 
for participation according to applicable laws, regulations, guidelines, and policies protecting privacy and 

 
Version 12, 15 May 2020, with Amendment 12  
 
confidentiality, and not until after a HIPAA Waiver of Authorization is approved by the IRB and R&D 
Committees. Following IRB and R&D Committee approvals, recruitment will be initiated among patients in the 
DVAMC Pain Clinic. If accrual milestones are not met, we will expand our search for eligible patients to include 
the DVAMC Rehabilitative Medicine and Rheumatology departments. 
 
A waiver for release of protected health information will be requested to obtain the name, address, phone 
number, and screening information for patients with scheduled appointments in the Pain Clinic, or 
Rehabilitative Medicine or Rheumatology departments. The use and destruction of this data will follow the 
policies and procedures of VA privacy regulations.  After receiving the authorization, the recruitment plan 
described in Section 2.1 will be used. Confidentiality will be maintained to the fullest extent allowed by law. 
Only authorized personnel will have access to research data. Participants will receive a unique study ID 
number that will be used to track all data. The cross-reference file linking subject identities to the study ID 
number will be maintained in a locked file cabinet or password-protected VA computer by the Co-PIs or a 
designated staff member. Psychophysiological data collected by sensors will be stored electronically without 
any identifying information. Analysis and interpretation of data files also will be conducted without the use of 
personal identifying information. Questionnaire data will not have identifying information on the forms. Once 
collated, coded data will be entered into password-protected VA computer for further analysis and processing.  
 
If patients exhibit excessive anxiety, research staff will check with the patient to ascertain if data collection 
needs to be temporarily discontinued or rescheduled. Adverse physical or psychological events sometimes 
occur even if they are not be directly related to the study protocol. If a participant experiences a major or minor 
physical or psychological adverse event while enrolled in this study, supportive measures described in the 
section of the consent form labeled ‘possible risks’ in will be implemented. The Co-PIs will be contacted 
immediately. The PI will consult with clinical support staff to refer the patient for appropriate follow-up care, if 
needed..  
Adverse Event Reporting. If patients develop a severe affective disorder or psychosis, these will be considered 
a serious adverse experience. The PIs will send an adverse events report to the VA IRB as soon as they are 
apprised that an adverse event has occurred. If the VA IRB or either of the PI’s recommend that the study be 
temporarily or permanently suspended, this action will be reported to the VA grant program officer or director. 
 
Data and Safety Monitoring. The PIs and research staff will routinely monitor the progress of the trial and the 
safety of participants. The PIs, in collaboration with study staff, will check accrual and attrition on a weekly 
basis. Subject eligibility forms will be completed on all participants to assure appropriate subjects are recruited. 
None of the study procedures are considered invasive and the risks to subjects are expected to be minimal. 
Subjects will be informed that the experimental protocol will be provided in addition to their usual care, will not 
affect their treatment as usual, and no other intervention is available as an alternative intervention. HRV-B 
training is a simple, non-invasive procedure and no major adverse reactions to the protocol are anticipated. 
The proposed procedures have been widely used; no adverse events were experienced by Dr.Burch in 
previous studies using HRV-B or actigraphy. Questionnaire data will be collected from the participants by face-
to-face interview using validated instruments and questions that are similar to those that might be encountered 
in a clinical setting. Participants will be informed that if they don’t want to answer a question, they can skip it. 
An actiwatch is similar in size, weight, and configuration as a wristwatch. Saliva collection also is simple and 
non-invasive. Saliva biospecimens will be labeled in a manner that ensures participant confidentiality without 
personal identifiers. The participants will be fully informed of the procedures, including notification that their 
medical records will be examined. Participants will voluntarily sign an Informed Consent document and HIPAA 
Authorization approved by the IRB before participation begins. 
 
We are planning a follow-up HRV-B session plus questionnaire and cognitive evaluation at 4 weeks and 8 
weeks post-training (Figure 7). Subjects will be told that they may contact either PI for any reason for 6 months 
after this final follow-up session. Subjects will be encouraged to make contact during this 6 month post-follow-
up period if they have questions, or if any problem arises that the subject believes may be related to this 
research. If, during the course of the protocol, any adverse reaction develops or a subject wishes to terminate 

 
Version 12, 15 May 2020, with Amendment 12  
 
his or her participation, the assigned treatment will be stopped. We will encourage any patients who have their 
assigned treatment stopped to maintain attendance at their training visits even without treatment, and attempt 
to collect the evaluation data on the appropriate schedule. Follow-up would be stopped if the subject advises 
us that he or she wishes to terminate all participation in the research.  During the consent process, the subject 
will be told that cessation of follow-up can be voluntarily obtained by signing a form requesting that no further 
contact be made. They also will be informed that termination of their participation will not impact their usual 
care at the DVAMC.  
 
The DVAMC uses a Research Compliance Officer (RCO) as the clinical trial monitor who performs audits of 
consent forms, reporting and regulatory compliance, and documentation according to Standard Operating 
Procedures of the VA Office of Research and Development, and the local DVAMC Research office. The 
monitor reports separately from the IRB structure. The monitor will also perform random audits on any trial 
including the proposed study. 
 
Data Accuracy and Protocol Compliance. Subject eligibility forms will be completed on all subjects to assure 
appropriate subjects are recruited. Biofeedback Certification Institute of America (BCIA) certification of training 
and skill in HRV-B or its equivalent will be required of the HRV-B professional. This research will follow the 
HRV-B training protocol adopted by the BCIA. Protocol compliance integrity will be monitored by supervision 
and communication between the biofeedback professional, research staff, and the Co-PIs. 
 
 
 

 
Version 12, 15 May 2020, with Amendment 12  
 
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