Behavioral Studies of Cardiovascular Disease
Phase: NA
Conditions: Cardiovascular Diseases
Sponsor: University of Pittsburgh

 
 
 
 
 
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Exercise, Brain, and Cardiovascular Health (eBACH)  
Approved Protocol 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NCT number: NCT03841669 
Last Updated: September 2nd, 2021 
 

 
 
 
 
 
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Table of Contents 
1.0 
SUMMARY: ............................................................................................................ 3 
1.1 
BACKGROUND AND RATIONALE: ................................................................... 4 
1.2 
INNOVATION: ...................................................................................................... 4 
1.3  
PRELIMINARY DATA: ......................................................................................... 5 
1.4  
STATISTICAL ANALYSIS: .................................................................................. 6 
1.5 
PROCEDURES: ................................................................................................... 10 
1.5.1  
SCREENING: .................................................................................................... 10 
1.5.2 
INCLUSION AND EXCLUSION CRITERIA: ................................................... 11 
1.5.3 
BASELINE TESTING: ....................................................................................... 14 
1.5.4 
POST RANDOMIZATION TASKS: ................................................................... 21 
1.5.5 
TIME POINT 2 (6 months): ............................................................................... 21 
1.5.6 
TIME POINT 3 (12 months – post intervention): .............................................. 21 
1.6 
RANDOMIZATION: ............................................................................................ 21 
1.7 
TARGETED ENROLLMENT TABLE ............................................................... 21 
1.8  
INTERVENTION: ................................................................................................ 22 
1.9 
PARTICIPANT SAFETY: ................................................................................... 24 
1.10 
DATA MANAGEMENT: ..................................................................................... 25 
1.11 
QUALITY CONTROL: ......................................................................................... 26 
1.11.1   MRI QUALITY CONTROL: ................................................................................. 26 
1.11.2  ACTIGRAPHY, FITNESS, EMA, CARDIOVASCULAR QUALITY CONTROL: .. 26 
1.12 
RESOURCE SHARING: ..................................................................................... 27 
1.12.1 
DISSEMINATION PLAN: ................................................................................. 27 
1.12.2 
DATA SHARING: .............................................................................................. 27 
1.12.3 
DATA SHARING AGREEMENT: ...................................................................... 28 
1.13 
REFERENCES: ................................................................................................... 29 
 
 
 
 

 
 
 
 
 
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1.0 
SUMMARY: 
Cumulative epidemiological evidence indicates that physical inactivity confers risk for 
cardiovascular disease (CVD)1, and that current physical activity (PA) guidelines 
(prescribed at >150 minutes per week of moderate to vigorous intensity aerobic PA) can 
reduce CVD morbidity and mortality2,3. The biological and behavioral pathways by 
which PA may reduce CVD risk is still unclear4. In line with the P01 focus on 
neurobiology of CVD risk, we are conducting a 12-month PA intervention with 150 
midlife and inactive adults randomized to: (1) 150 minutes per week of moderate to 
vigorous intensity exercise (i.e., brisk walking; N=75); or (2) a PA and health 
information group where we will provide cardiovascular information and active daily 
monitoring (N=75). A comprehensive battery of cardiorespiratory fitness (CRF), 
cardiovascular health, physiological biomarkers such as endothelial function, and 
magnetic resonance imaging (MRI) measures will be assessed at baseline, after 6-months, 
and again after completion of the intervention.   
We have assembled a highly creative, productive, and interdisciplinary team with 
a long history of collaboration and experience conducting exercise interventions to test 
the following aims:  
Aim 1: To determine the Neurobiology of Exercise and Biological CVD risk 
factors:  
(1A) Body-to-Brain hypothesis: Exercise-induced changes in peripheral markers 
of CVD risk (i.e., insulin resistance, pulse wave velocity, CRF, oxidative stress, 
biomarkers of endothelial function) will precede and partly explain (statistically mediate) 
exercise-induced changes in functional and structural features of some areas within 
visceral control circuits (prefrontal cortex [PFC], anterior cingulate cortex [ACC], insula, 
hippocampus, and amygdala).  
(1B) Brain-to-Body hypothesis: Exercise-induced changes in functional and 
structural features of some areas within visceral control circuits precede and partly 
explain (statistically mediate) consequent changes in peripheral autonomic and 
neuroendocrine mediators of CVD risk that are under neural regulation, including 
baroreflex sensitivity, heart rate variability, glucocorticoid control.  
Aim 2: To determine the Neurobiology of Exercise and Stress- and Affect-related 
CVD risk factors:  
(2A) Stress-related parameters of CVD risk: Exercise will induce changes in 
visceral control areas engaged by an fMRI stress battery, and these changes will partly 
explain exercise-induced reductions in cardiovascular stress reactivity in daily life.  
(2B) Affect-related parameters of CVD risk: Exercise will induce changes in 
visceral control areas engaged by an fMRI emotion processing and regulation paradigm 
in synergy with Project 1 (P1), and these changes will partly explain exercise-induced 
improvements in positive and negative affect measured in daily life by EMA and by 
conventional self-report instruments in synergy with Project 2 (P2).  

 
 
 
 
 
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1.1 
BACKGROUND AND RATIONALE: 
PA is one of the most critical behaviors to reduce risk for many chronic diseases, 
including some cancers (e.g., breast cancer), cardiometabolic conditions (e.g., Type II 
diabetes), and atherosclerotic cardiovascular disease (CVD)4-6. Moreover, PA has 
beneficial effects on a range of proximal mediators and markers of chronic disease risk—
especially CVD risk. These include blood pressure (BP), cardiac autonomic control, 
inflammation, glucose regulation, adiposity, and lipid levels. In patient samples, clinical 
trials show that PA accelerates recovery from myocardial infarction8, coronary bypass 
surgery and congestive heart failure9, improves quality-of-life, and reduces risk for new 
CVD events and all-cause mortality10. Further, cardiac patients who improve their fitness 
show a 20% reduced mortality rate over an 11-year follow-up period11. Based on an 
expansive body of evidence, the American Heart Association (AHA) has identified CRF 
as an important ‘vital sign’, and it has recommended more frequent assessments and 
management of fitness by health professionals12,13. Finally, physical inactivity is 
estimated to account for >350,000 deaths per year in the USA alone, with an additional 
3.2 million annual deaths globally14. In view of clear scientific evidence on PA and 
health, many international organizations have nearly identical recommendations for 
people to engage in PA. The most recent were established in 2018, advising >150 min of 
moderate intensity exercise, or at least 75 min of vigorous exercise, per week to improve 
cardiovascular health and to reduce risks for premature mortality2. Less widely 
appreciated by health care professionals, the public, and policy makers are the broad 
benefits of PA on the brain. These benefits translate into brain-based improvements in 
cognitive, affective, stress-buffering, and self-regulatory behaviors 5,7. Indeed, substantial 
human and animal evidence shows that the brain exhibits appreciable structural and 
functional plasticity (i.e., change) from PA. As a result of this evidence, many 
organizations that influence public policy have developed campaigns to educate the 
public and health care professionals on the brain benefits of PA. For example, the NHLBI 
and AHA note mood improvements and reductions in psychological distress as 
established benefits of engaging in PA 15. In support of these health campaigns, Barnes & 
Yaffe19 further reported that >1.1 million Alzheimer’s cases in the USA are potentially 
attributable to physical inactivity. Noteworthy here is that Alzheimer’s and other forms of 
dementia are highly co-morbid with CVD, and they are often predicted by CVD risk 
factors that are affected by PA (e.g., hypertension)16-21. Moreover, exercise reduces 
aspects of negative affect and depressive symptoms that associate with CVD risk22-24. 
Finally, there is preliminary evidence to suggest stress-buffering effects of PA and 
cardiorespiratory fitness at the level of physiology and behavior, which may define 
additional pathways to reduce CVD risk. What is not known, however, is whether the 
beneficial effects of exercise on the brain extend beyond emergent cognitive, affective, 
and stress-buffering processes to also include beneficial effects on physiological and 
systemic mediators of CVD risk that are regulated by the brain. If so, such effects may 
help to more precisely define and refine novel and brain-based targets for intervention 
and prevention efforts.  
1.2 
INNOVATION: 
We have assembled a productive team with a history of collaboration in exercise 
interventions. eBACH questions are unique in the field, potentially transformative, and 
relevant to both scientists and health professionals.   

 
 
 
 
 
5 
• Body-to-brain pathways linking physiology and exercise. For the first time, we 
will test whether exercise-induced changes in peripheral markers of CVD risk explain 
changes in brain plasticity. 
• Brain-to-body pathways linking exercise and physiology. We will test the novel 
hypothesis that the effects of exercise on brain circuits supporting visceral control 
account for changes in autonomic, hemodynamic, and neuroendocrine mediators of 
CVD risk. 
• Dense repeated measurement of exercise and neural-CVD risk associations. For 
the first time in a randomized exercise intervention, we will collect 3 measurement 
points of high-resolution neuroimaging data and peripheral mediators and markers of 
CVD risks. This allows us to more accurately model trajectories of change for both 
neuroimaging and CVD risk related measures, enabling us to move beyond traditional 
correlation, observational, and briefer wave assessments of these associations.  
• Statistical modeling of exercise and CVD risk. We have the expertise to apply 
cutting-edge machine learning approaches to neuroimaging, behavioral, and 
peripheral biomarkers to develop a more comprehensive and realistic model of the 
associations between exercise and its associations with peripheral physiology, risk 
factors for CVD, and brain plasticity.  
• Neurobiology of exercise and emotion and stress linked to CVD risk. We will 
examine how exercise alters stress physiology and affective CVD risk correlates via 
changes to brain circuits. 
 
1.3  
PRELIMINARY DATA: 
1. Peripheral mediators and markers of CVD risk and PA: In the AHAB2 
registry, we tested associations between markers and mediators of CVD risk and PA after 
control for age and sex. In AHAB2, energy expenditure (assessed over ~5 days by 
actigraphy) was negatively correlated with mean carotid intima-media thickness (p = 
0.017, N = 437) and an aggregate metabolic risk score (p < 0.001, N = 414). Also, energy 
expenditure was positively correlated with an index of cardiac vagal control derived from 
high-frequency heart rate variability (HF-HRV) and root mean square of successive 
differences (RMSSD) measured during paced and unpaced breathing epochs (p < 0.001, 
N = 432). Moreover, greater energy expenditure correlated with lower interleukin 6 (IL-
6) and C-reactive protein (CRP) (ps < 0.001, Ns = 453 and 454). The cardiac vagal 
control index also negatively correlated with IL-6 and CRP (p’s < 0.005, Ns = 448 and 
449). In line with our aims, energy expenditure was positively correlated with bilateral 
hippocampal volume after additional control for intra-cranial volume (ICV) (p = 0.024, N 
= 425). Finally, the cardiac vagal control index was positively correlated with volumes of 
the ACC, amygdala, and hippocampus (ps < 0.05).  Implications for this project: These 
results show associations between PA and peripheral markers and mediators of CVD risk, 
and that these associations may be linked to markers of brain plasticity and health. What 
is unknown, and what is tested here, is whether such cross-sectional associations between 
CVD risk markers, PA, and brain health are causally linked in the context of an 
experimental manipulation of PA.   
2. PA, stress, and affect: In brief, we replicate several observations from the 
literature on associations between PA and fitness with affect, and we provide the first 

 
 
 
 
 
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evidence that PA relates to blunted stressor-evoked BP reactivity in daily life. We also 
report the first associations to our knowledge on PA and individual differences in neural 
activity during the processing of negative emotional stimuli, as well as during the 
regulation of negative emotions. In aggregate, these initial findings support the 
integration of the fMRI stressor and emotion task batteries, as well as ambulatory (ABP) 
and ecological monetary assessment (EMA) methods. 
3. Exercise and brain health: Our research group has published >170 papers on 
the effects of exercise and fitness on brain morphology, white matter integrity, resting 
state connectivity, and task-evoked functional MRI activity. But, none of these have 
specifically addressed the questions at issue here. Evidence from our work and others 
reliably demonstrates that PA and CRF are related to several metrics of brain health 
across the lifespan in clinical and healthy samples. For example, we found in children 
(N=35/group) that exercise is associated with changes in task-evoked fMRI29, cerebral 
blood flow in the hippocampus30, and increased hippocampal volume31. Similar effects of 
exercise are seen in young adulthood and mid-life. For example, we found in a 
randomized trial of PA (N=125) in adults between 18-50 yrs that 12-months of PA 
increased cerebral blood flow in the ACC, insula, and PFC. Increases in hippocampal and 
PFC volume is also seen in young adults after 6-months of aerobic exercise32. Similar 
positive effects of PA have been reported in mid-life adults with T1 diabetes33, in 
multiple sclerosis patients34, and in women with breast cancer35. Positive effects of PA on 
brain health are often observed in late-life. For example, in 120 older adults (60-80 yrs; 
N=60/group), moderate intensity exercise increased hippocampal volume. Exercise also 
increased volume of the PFC and ACC in 59 (N=~28/group) older adults26. In 879 older 
adults, greater energy expenditure was associated with greater volume of the 
hippocampus, ACC, and PFC36. In addition to brain morphology, changes in CRF 
resulting from an exercise intervention were correlated with increased white matter 
integrity in PFC and temporal lobes in 70 older adults28 and also in two larger samples 
(N=113; N=154). Our results also extend to resting-state connectivity27,37,38 and task-
evoked activation39. For example, in 65 older adults exercise increased functional 
connectivity27. Overall, this rapidly growing literature indicates that PA, CRF, and 
aerobic exercise in particular, is effective for improving several metrics of brain health in 
visceral control circuits and that these effects occur across the lifespan in diverse 
samples. 
1.4  
STATISTICAL ANALYSIS: 
 
1.4.1.  POWER:  Our proposed intervention is much larger and much longer than 
other similar well-controlled randomized PA interventions, especially those that 
include neuroimaging. Prior studies are typically <6 months in duration with 
N<25/group. Thus, our intervention with 3 measurement points (0, 6, 12 months) 
with N=150 (75/group) far exceeds other comparable studies in this area. Well-
controlled RCTs typically have smaller sample sizes than traditional observational 
studies because of the increased power obtained through the within-subject 
analytical approach. Indeed, the number of assessments (450) is similar to that of 
Projects 1 and 2. Here we describe the statistical basis for our proposed design 
using an estimated sample size after 20% attrition (N=60/group). Brain 

 
 
 
 
 
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volumetrics: Our prior 6-12 month PA interventions in children, young adults, 
and older adults have used between 2026 and 6040 subjects per group with 
moderate-sized effects (Cohen’s d) ranging between 0.30 and 0.50. Cross-
sectional research has supported these numbers with studies reporting significant 
effects of PA on brain volume with only 32 subjects41 and slightly larger effect 
sizes between 0.50 – 0.60. Even if we estimate a rather conservative effect size of 
0.30, 60 participants per group is sufficient to detect differences in brain volume 
at 80% power. Diffusion imaging: Changes in white matter have been detected in 
PA interventions in as little as 6-months with only 50 subjects26. In a 12-month 
exercise intervention, we found increased FA with only 35 subjects in each group 
with an estimated effect size of 0.7028. Fewer results have been reported in mid-
life, but in children, effects of PA on white matter have been reported with 
N=25/group. Based on an estimated effect size of 0.75, we should have 90% 
power with 60 participants per group to test our main hypotheses as well as 
enough power for exploratory analyses. Connectivity: With only 65 people, we 
have shown that a 12-month exercise intervention is effective at enhancing 
functional connectivity in prefrontal-inhibitory networks27. The difference 
between the groups for PFC connectivity was .15 with a standard deviation of .10 
suggesting an effect size of 1.5 (Cohen’s d). In young adults, we report similar 
effects with N=50 with increased functional connectivity between the anterior 
hippocampus and ACC. Therefore, similar to the effect sizes and power for the 
other neuroimaging measures, 60 participants per group should be sufficient for 
detecting changes as a function of the intervention with 95% power. Stress 
reactivity and affect:  In preliminary data from the AHAB2 cohort, greater PA 
was associated with reduced daily life stress reactivity measured by strain (r=.23) 
and conflict (r=.25). In a 12-week exercise intervention in young adults with 
major depression scores on the Montgomery Asberg Depression Rating Scale 
significantly decreased from 21.8 to 4.25, and the magnitude of this decrease was 
correlated with increases in fitness (r=-.39). Based on these effect size data, 60 
participants per group would provide us with >90% power to detect an effect on 
stress and affect measures. Thus, we estimate adequate power to test Aim 2. CVD 
markers: Prior randomized exercise interventions examining some of the same 
CVD risk markers proposed here (e.g., baroreflex sensitivity [BRS]) are typically 
between 4-52 weeks in duration with N= ~10 and 15031.  Despite this 
heterogeneity, meta-analyses of exercise interventions show moderate-to-large 
effects on many of these outcomes (Cohen’s d=.60). For example, a meta-analysis 
of 30 exercise interventions on multiple CVD risk markers reported an average 18 
week duration with ~50 subjects in each study (~25/group)25. In this meta-
analysis, CRF increased by 3.04 mL/kg/min with a Cohen’s d of 0.58. We expect 
larger effect sizes here because durations of ~1-year demonstrate larger changes 
in these metrics42. Thus, based on prior studies and meta-analyses, we are 
sufficiently powered with N=150 to test for exercise-induced changes in CVD 
markers. Mediation: Using established empirical estimates of sample sizes needed 
for .80 power in bias-corrected bootstrapping analyses with moderate sized effects 
in both alpha and beta, it is estimated that 115 participants are needed43. Thus, 
based on the above effect sizes for both brain and CVD markers, we are 

 
 
 
 
 
8 
sufficiently powered to test our hypotheses using structural equation modeling 
even after attrition. In sum, our planned sample size of 150 (N=75 per group) will 
be sufficient for allowing us to test our primary hypotheses. 
 
1.4.2. PRELIMINARY STEPS: Prior to hypothesis testing, all data will be 
examined to determine: 1) frequency distributions for missing data and out-of-
range values; 2) normality and internal consistency of subscales; and, 3) 
association between variables that may be highly correlated to guard against 
multicolinearity, which would inflate standard errors and make estimation 
unstable. We will also examine the distribution 
of factors between groups to assess whether 
covariate-adjustment is required. Missing data: 
All outcomes will be tested using an intent-to-
treat (ITT) framework. Sensitivity will be 
explored using adherence to the intervention 
(e.g., per protocol). We will report 
reasons for dropouts and explore missing data 
mechanisms. To examine whether missingness 
is unrelated to other observed and unobserved 
measurements (missing completely at random, 
MCAR) or to the observed measurements only 
(missing at random, MAR), testing for 
completely random dropouts will be carried 
out. Additional sensitivity analyses test for 
informative dropouts. We will apply pattern-
mixture models44 by stratifying our data by dropout patterns and fitting separate 
regression models to strata.   
 
1.4.3.  PRIMARY OUTCOMES:  Before testing the specific aims related to 
mediation we will test whether the intervention modifies proximal endpoints that 
will serve as the primary outcomes of the study.  Thus, the primary endpoints will 
be 12-month change scores resulting from three different measurements: (1) 
hippocampal volume, (2) mean systolic blood pressure reactivity averaged from 
Stroop and Multi-Source Interference Task (MSIT) stressors during fMRI, and (3) 
heart rate variability.  More specifically, we are predicting Time x Group 
interaction terms such that the exercise arm would change more than the control 
arm for these measures. Changes in scores of other measures including EMA-
derived measures of daily life positive and negative affect, BRS, pulse wave 
velocity [PWV], vasodilation, CRF, resting state brain connectivity, and cortical 
thickness will be considered secondary and tertiary outcomes. Further, results 
from the structural equation modeling will be considered secondary outcomes, but 
will be testing the specific aims of the project. 
 We propose to test our primary hypotheses using a general linear mixed model 
(GLMM) approach because we will have three assessments for each of our 
measures over the course of the trial (baseline, 6-months, 12-months). The 
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a 
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XT3 
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Figure 1. Diagram of exemplar 
full longitudinal mediational 
model. Paths involved in 
estimating longitudinal stability 
and residualized change are in 
black, paths involved in mediation 
are in red, and non-essential 
parameters are grayed out for 
legibility. 

 
 
 
 
 
9 
GLMM model will include both random intercept and random slope for 
individual participants and a treatment-by-time interaction as a fixed effect. This 
technique will allow us to model the changes in the scores as a function of both 
time and group while also including other potentially confounding variables in the 
model. GLMM modeling of repeated assessments is a popular approach in clinical 
and longitudinal studies because it allows for random changes in measurement 
error and takes into account both within and between subject correlations and 
estimates the average growth curve across subjects and subject-specific growth 
curves. 
 
1.4.3  MEDIATION:  Aim 1 has two hypotheses, (a) to test whether the 
peripheral effects of exercise mediate brain effects, and (b) whether the effects of 
exercise on the brain mediate the beneficial effects of exercise on peripheral 
mediators of CVD risk. We propose to test these hypotheses using longitudinal 
mediation models in a structural equation modeling (SEM) framework. Aim 1A: 
In this model, the exercise groups (X) are proposed to influence visceral control 
circuits (Y) via changes in peripheral pathways (M) including insulin resistance, 
oxidative stress, endothelial dysfunction, and CRF. We will first examine whether 
brain or these peripheral CVD pathways can be summarized as latent variables to 
control for measurement error. When using latent variables, the modeling will 
proceed by first establishing well-fitting measurement models, and testing for 
measurement invariance across waves. To test the hypothesis proposed in Aim 
1A, analyses will be conducted both on a voxel-wise basis (e.g., using the 
BRAVO Toolbox (https://sites.google.com/site/bravotoolbox/)) or using extracted 
data from SPM, FSL, or FreeSurfer, depending on the imaging modality and 
analysis. In the latter case, we will use a theory-driven approach by selecting 
values from anatomical or functionally-defined regions-of-interest (ROI) on 
visceral control regions. Each imaging modality will be analyzed in separate 
models unless we have a theoretical or empirical basis for including multiple 
imaging metrics in a single model. We will use standard cluster-based and FDR 
corrections for multiple comparisons for all voxel-wise analyses and the Brain 
Connectivity Toolbox for graph theory metrics (i.e., segregation). Although 
directional hypotheses have been posited for our mediation analyses, a more 
conservative significance will be set at .01 to limit inflation of type 1 error. Global 
and incremental model fit will be verified using multiple fit-indices (chi- square, 
RMSEA, CFI, TLI, SRMR). With three waves of data, we will use full 
longitudinal mediation to test our hypotheses. Full longitudinal mediation allows 
us to test the effect of initial standing on the hypothesized predictor (X – Exercise 
group) at T1 on change in the mediator (M – peripheral CVD markers) from T1 to 
T2, controlling for initial standing on M, and the effect of the mediator (M– 
peripheral CVD measures) at T2 on the change in the outcome (Y – change in 
brain measure) from T2 to T3, controlling for initial value of the outcome. 
Mediation will be tested by calculating the indirect effect of paths that are 
consistent with longitudinal mediation (i.e., paths a and b in Fig. 1). Here the 
estimate a*b provides a measure of the longitudinal mediated effect because it 
represents the effect of X at T1 on residualized change in M (path a), and M at T2 

 
 
 
 
 
10 
on residualized change in Y (path b).  Significance of the mediation (i.e., indirect) 
effect will be examined using techniques that account for non-normality in the 
parameter distribution, namely bias corrected bootstrapping-based approaches. A 
benefit of full longitudinal mediation models is that the direction of the paths can 
be reversed (Y->M->X) to test for the opposite causal direction, and bidirectional 
effects can similarly be tested. We will test for these possibilities by comparing 
models with paths reversed and allowing bi-directional effects. Finally, we aim to 
explore the role of other potential mediators (i.e., sleep) and moderators (e.g., 
gender) on the direction and strength of associations. Aim 1B: We will proceed 
with similar approaches described above for Aim 1A including using a voxel-wise 
approach and a theory-driven approach by selecting anatomical or functionally-
defined ROIs on visceral control circuits or the white matter pathways that 
connect them. Importantly, there may be regional dissociations between the brain 
regions identified in Aim 1A and those identified in Aim 1B. Using voxel-wise 
analyses, we will be able to adjudicate the regional specificity or overlap between 
the regions identified in Aim 1A with those of Aim 1B. Using these procedures, 
we will also be able to determine the specificity of particular effects (e.g., regions 
associated with BRS but not with HRV) or differences across imaging metrics 
(e.g., differences of effect between functional connectivity, cortical thickness, 
white matter). With three waves of data, we will use full longitudinal mediation to 
test our hypotheses that X (Exercise group) influences BRS, HRV, and 
glucocorticoid control (Y) via its effects on brain plasticity on visceral control 
circuits (M). The description of the SEM modeling approach for Aim 1A will be 
applied to test mediation for Aim 1B. To test Aim 2A & B: The analytical plans 
for testing Aim 2A and 2B along the lines depicted in the heuristic model of 
Figure 1 will proceed like that described above for Aim 1A and B. That is, we 
will test how the effect of the hypothesized predictor (X – Exercise group) 
influences the change in the mediator (M – visceral control circuits), on stress 
reactivity and affect metrics that are measured in Projects 1-2 (e.g., EMA and 
ABP measures identical to those used in NOAH). Similar to Aim 1, we will be 
able to model opposite causal directions and bidirectional effects by reversing the 
paths in the model and will be able to determine specificity of particular effects 
(e.g., regions associated with emotion regulation, but not daily life stress 
reactivity) or differences across imaging metrics (e.g., differences of effect 
between functional connectivity, cortical thickness, white matter). As above, we 
will also test other potential mediators (i.e., sleep) and moderators (e.g., gender) 
on the direction and strength of associations. 
 
1.5 
PROCEDURES: 
Before the collection of baseline data, all staff will have had ethics training and 
appropriate certification of research training modules.   
1.5.1  SCREENING: 
During the UCSR telephone screening interview, numerous medical history 
questions will be asked to verify that the potential participant meets inclusion 
criteria as no medical record retrieval is being used. Information about safety to 
exercise will be obtained over the phone (i.e., history of falls) to ensure that 

 
 
 
 
 
11 
exercise will not be dangerous for the participant. Once deemed eligible, this 
participant’s contact information will be sent to an eBACH staff member who will 
conduct a secondary screen specifically for physical activity and MRI safety 
eligibility. Information about the participant’s current exercise status will be 
collected and participants that engage in more than 75 minutes of structured 
exercise per week will be considered ineligible. Information regarding metal in 
the body for MRI safety will obtained, including information about any metallic 
objects in their body.  If a participant is deemed unsafe to exercise based on the 
physical activity readiness questionnaire, the participant will be required to have 
prior approval by their primary care physician before being admitted into the 
exercise program.    
 
1.5.2 INCLUSION AND EXCLUSION CRITERIA: 
Inclusion Criteria:  
• Age – individuals aged 28-56 years old when signing the consent 
form.   
• Gender & Ethnicity – men and women are both eligible to 
participate. We expect the study population to be approximately 50% 
women. All ethnic groups are eligible for the study. We expect the 
study population to be approximately 30% minorities.  
• Ambulation – study participants must be able to walk without pain or 
use of an assisted walking device. This will be determined during the 
initial phone screen. Ambulation will also be assessed in person as the 
potential subject must be able to complete the VO2 Max test on a 
treadmill.  
• Physical Activity and Fitness Level – all participants must report 
engaging in less than 75 minutes per week of exercise and have a 
VO2max percentile less than 75 based on the ACSM Guidelines for 
Exercise Testing (10th edition). 
• Residency - Since the intervention is 12 months in duration, all 
potential participants must reside in the Pittsburgh area and plan on 
residing in the area for at least one year after randomization. It is 
recommended that the study coordinator during the second screen 
assess potential participants’ distance from the exercising sites since 
the study requires 2 visits per week. The further a participant is from 
an intervention site, the higher the probability is for the person to be 
unable to adhere to the protocol.   
• Reliable transportation:  Participants must have reliable 
transportation to complete the outcome measures and the intervention 
requirements.  
• Willingness to be randomized - To be eligible, all potential study 
participants must be willing to be randomized to either of the 
intervention arms. Individuals that insist on participating only if they 
can choose which arm they belong to should not be enrolled. 
 
Exclusion Criteria:  

 
 
 
 
 
12 
• Medication Use:  
A.  Reported use of the following medications on one or more 
occasions in the past 14 days constitutes grounds for exclusion: 
 
    
1. Antihypertensive or cardiac medications (diuretics, beta 
blockers, calcium channel blockers, ACE inhibitor/ARB, 
cardiac glycosides, central sympatholytic HTN drugs, anti-
arrhythmic drugs, vasodilator drugs, other cardiac drugs). 
 
 
 
2. Anticonvulsant medications 
 
 
 
3. Antiparkinson medications 
 
 
 
4. Protease inhibitors or other Anti-HIV medications 
 
 
 
5. Medications for the treatment of mania 
 
6. All other centrally active or psychotropic medications 
excluding anti-anxiety, antidepressant, or 
antipsychotic/tranquilizer medications. 
 
 
 
7. Insulin 
 
 
 
8. Chemotherapy 
 
9. Immunosuppressants and related biological agents 
(Imuran, methotrexate, and cyclophosphamide) 
10. Prescription weight loss medications and ephedrine 
OTC  
 
B. Reported use of the following medications on a “regular” basis 
is grounds for exclusion. For this purpose, “regular use” is defined 
as reporting that the prescribed medication was taken 7 or more 
days in the past 14 days. Individuals who take these medications, 
but less frequently than 7 out of the past 14 days are not excluded: 
 
 
 
1. Anti-anxiety medications (Ativan, valium, Xanax) 
 
 
 
2. Sleep medications (e.g., trazadone at bedtime for sleep) 
 
 
 
3. Asthma oral medications 
 
 
 
4. Asthma/allergy inhalants (alupent, albuterol) 
 
 
 
5. Anti-depressant medications 
 
 
 
6. Antipsychotic/tranquilizer medications 
 
 
 
7. Glucocorticoids (oral; prednisone, cortisol) 
 
 
 
8. Medical marijuana 
C. Reported use of more than 2 non-insulin medications for 
diabetes on a “regular” basis is grounds for exclusion. For this 
purpose, reported use of combination medications, involving two 
or more non-insulin medications for diabetes in a single pill, is 
counted as 2 separate medications and would be grounds for 
exclusion. 
D. A person who reports that he or she was once on a disallowed 
medication, but has discontinued this medication for at least a 
month or longer and is otherwise eligible, is allowed to participate 
in the study.  
• Substance use exclusions: 
A.  Anyone reporting 35 or more alcoholic drinks in the last 7 days 
is excluded. 

 
 
 
 
 
13 
 
B. Anyone reporting consumption of 6 or more alcoholic drinks on 
3 or more occasions in the past 7 days is excluded. 
C. Anyone reporting a score of 8 or more on the 10-item AUDIT 
scale (indicating harmful or hazardous use of alcohol) is excluded. 
D. Anyone reporting use of illicit drugs on 7 or more days   
 
 
in the past 2 weeks is excluded. 
• Medical conditions that are disallowed are: 
 
A. Self-reported prior heart attack, stroke, bypass surgery, 
angioplasty, congestive heart failure, arrhythmia (cardiac rhythm 
problems). 
 
 
B. Hypertension (SBP/DBP > 160/and/or >100 mmHg). 
 
C.  Cancer (treatment in last 12 months, allowance for non-
melanoma skin cancer) -  potential participants who have received 
treatment in the last 12 months for cancer, including radiation or 
chemotherapy will be excluded from the study. 
 
D. Liver disease - any person who has had hepatitis B or C, liver 
failure or Cirrhosis will be excluded from the study. 
 
E. Kidney disease - potential participants who indicated they have 
chronic kidney failure, have undergone dialysis, or have had a 
kidney transplant will not be eligible to participate. 
F.  Type I diabetes or insulin dependent Type II diabetes - Type 2 
diabetics if they are taking insulin or 3 or more diabetes 
medications. Single injections that contain 2+ drugs count as 2 
medications.  
 
G. Self-reported history of a major neurological disorder or brain 
injury resulting in ongoing symptoms or cognitive impairment 
(e.g., multiple sclerosis, cerebral palsy, major head injury) 
 
H. Self-reported chronic psychotic illness (schizophrenia, bipolar 
disorder).  
 
I.  Lung disease requiring drug treatment (asthma or allergy 
inhalers are not exclusionary unless they are used on a “regular 
basis”) 
 
 
J. Weight loss surgery within the past 5 years. 
• Other exclusionary criteria: 
 
A. Current pregnancy or plans to become pregnant over the next 
year – any woman who is pregnant via a pregnancy test at the 
baseline visit will be excluded from the study. A pregnancy test 
will also be given at the MRI visit and this will be repeated at 6-
months and 12-months. 
 
B. Those who make regular use of an assistive walking device are 
excluded. 
 
C. Those who are not fluent in English are excluded  (defined as 
having spoken and read English every day for <10 years). 
            
D. Those with a visual impairment that would prevent them from 
reading printed text or text on a computer screen, iPad, or other 
electronic device are excluded.  

 
 
 
 
 
14 
 
E.  Those with color blindness, who may not be able to distinguish 
colors on some of the tasks used in this study, are excluded. 
 
F. Those who have claustrophobia (fear of enclosed or confined 
spaces) are excluded. 
 
G. Those who have medical devices, implants or other metal 
objects in or on the body that cannot be removed and are 
incompatible with use of fMRI (for example, tattooed eyeliner) are 
excluded. 
 
 
H. Those who are unable to fit into an MRI scanner are excluded. 
 
I. Those who report working the night shift on a frequent basis 
(half or more of the hours worked in a full workday are between 
midnight and 8 am, and this has occurred more than 12 times 
during the past year) are excluded. 
 
J. Those without reliable access to a telephone throughout the day 
(home, work, or cell phone) are excluded. 
         
K. Those who are otherwise unable to meet the requirements of the 
study (for example, persons whose employment or personal 
situation will not permit momentary interruptions required for 
electronic diary and ambulatory data collection) are excluded.  
 
L.  Those unwilling to be randomized to one of the two groups will 
be excluded. 
 
M.  Individuals that self-report engaging in 75 minutes or more of 
structured physical activity per week will be excluded. This will be 
determined using a combination of questionnaire and interrogation 
by trained exercise staff. 
 
N.  Those that have cardiorespiratory fitness levels that are above 
the 75%ile based on ACSM criteria will be excluded. 
  
1.5.3 BASELINE TESTING: 
There are multiple outcome measurement sessions at baseline. There is an 8-week 
maximum window to complete these measures.  The 8-week window starts the 
day the informed consent is signed at session 1. Any measures or sessions 
collected outside of this window would not determine eligibility, but would be 
classified as a protocol deviation. Further, the order of these sessions and 
measures might be re-ordered based on availability and timing of available slots. 
 
Session 1: ELIGIBILITY AND FITNESS TESTING 
Before the session, participants will receive a reminder phone call and will be 
instructed to refrain from exercise or drinking alcohol after 9:00 pm the night 
before, no over-the-counter medications 12 hours prior to the appointment, no 
smoking, using nicotine, or consuming caffeine 3 hours before the visit. Informed 
consent will be obtained during the first session (~ 3 hours). Participants will have 
their blood pressure taken (clinical read) to re-verify eligibility. Measurement of 
the participants height and weight will be taken using a validated stadiometer. 
Participants will be asked to bring their medications in a bag for verification. 
Once the participant is deemed eligible, they will fill out several questionnaires 

 
 
 
 
 
15 
before undergoing a CRF test. Participants will complete a maximal VO2 test to 
assess CRF following a Modified Balke Protocol with an agreed upon speed 
between the participant and lead exercise physiologist. The session will first start 
by a warm-up session of 5 minutes followed by a blood-pressure reading. Only 
participants with safe blood pressure readings (>220/110) will be allowed to 
continue. The participant will walk on a motor-driven treadmill with constant 
speed and increments of the incline. The intensity is increased in two-minute 
stages. We will collect all of the exhaled air by having the individual wear a fitted 
facemask that collects the exhaled air which then travels through a tube to the 
analyzer. This assessment usually takes about 15 - 30 minutes to complete 
depending on the starting fitness level of the participant. These procedures are 
recommended by the American College of Sports Medicine for graded exercise 
testing. During the exercise test, heart rate is continuously monitored via ECG 
along with blood pressure readings and Rating of Perceived Exhaustion (RPE) 
every two minutes. When the participant reaches the endpoint goal of the exercise 
test (symptom limitation and/or volitional exhaustion), the facemask will be 
removed and they will undergo a four-minute active cool-down in which they will 
walk at a slower rate with zero incline grade. After these four minutes the 
participant will be helped off of the treadmill and will undergo a passive cool-
down session in which they are seated in a chair. The participant's heart rate and 
BP will be continuously monitored through the cool-down period every two 
minutes and the participant will not be allowed to leave until the vital signs return 
to baseline patterns (within a value of 20 mmHg of systolic and diastolic and 20 
beats per minute of heart rate relative to their baseline reading). 
 
Each maximal exercise test will be administered by a trained exercise physiologist 
and exercise technician. The exercise technician should be the staff member who 
records the required values of each stage and assists with test administration. The 
exercise physiologist should be the staff that leads the exercise test and provides 
verbal cues to the participant. The exercise testing will be used to measure 
changes in fitness levels after 6-months and 12-months. The room should be set at 
a comfortable temperature (recommended between 60 – 70 degrees Fahrenheit), 
have necessary fans in place, and have access to water for the participants.  The 
exercise physiologist will also be responsible for examining the ECG read-out and 
anyone displaying any questionable patterns will have the ECG read by a 
consulting cardiologist for the team for confirming safety or need for additional 
follow-up with the participants PCP or cardiologist.  Continued eligibility for the 
study will be dependent upon this consultation and follow-up. 
 
Also at this visit, participants will be fitted with a commercially available physical 
activity monitoring device (Actigraph Link) around the non-dominant hand wrist 
that will record objective physical activity data over the duration of approximately 
one week. The participants will be provided with detailed instructions regarding 
wearing of the device as well as the option to remove the device if it becomes 
problematic. Participants will complete a series of questionnaires at this visit. 
 

 
 
 
 
 
16 
Once the VO2max session is complete, participants will be given the NCI diet 
history questionnaire, via a link and study code that is unique to each participant. 
Participants will have the option to complete the questionnaire online at their 
home or via a paper based method to be returned to the eBACH staff.    
 
Session 2: EMA INFORMATION   
This visit will take approximately three hours and the entire session will occur at 
Sennott Square in Oakland. The visit entails a clinic blood pressure assessment, 
and therefore participants will follow the same pre-visit restrictions as visit 1 (no 
exercise or drinking alcohol after 9:00 pm the night before, no over-the-counter 
medications 12 hours prior, no food or drink (except water), no smoking, using 
nicotine, or consuming caffeine 3 hours before the visit). Participants will be 
notified of these restrictions by a phone call made by a research assistant at least 
one day prior to the visit. This call and all calls to the participant during the daily 
monitoring phase will be made from the study cell phone. 
 
Additionally, participants will complete several questionnaires assessing their 
physical and emotional well-being. 
 
At this visit participants will also receive training on how to use several 
ambulatory monitoring devices that they will carry with them during the 10-day 
daily monitoring phase of the study. These devices will measure physical activity 
and sleep (actigraphy wristband), ambulatory blood pressure (portable blood 
pressure device, belt, and cuff), and daily psychosocial stressors (Diary of 
Ambulatory States [DABS] completed on a smart phone). Participants will 
receive detailed instructions on how to use these monitoring devices, including an 
option for removal if any of them present a problem. Following the 10-day 
monitoring period, we will collect the devices, and after we download the data we 
will clean and sanitize them so that they can be used by other participants in the 
study. While participants wear the devices they will not be able to view any data.  
 
After the training session, participants will leave the lab practice using the 
monitoring equipment for the rest of the day and for the morning of the following 
day. Around late morning, a research assistant will call participants for their 15-
minute “Practice phone call,” in which they will be given assistance with any 
problems or questions that may have come up during the practice period.  
 
Following the practice phone call, participants will be instructed to begin the 10-
day monitoring phase. This phase consists of four “full” monitoring days (using 
all devices and completing all interviews) and six “partial” monitoring days 
(using only the actigraphy device and the electronic diary to complete a morning 
interview and an evening interview).  
 
On the “full” days, participants will have their blood pressure taken every hour 
they are awake. Following each blood pressure reading, they will complete a 2-5 
minute questionnaire (DABS) about their thoughts and feelings using the 

 
 
 
 
 
17 
electronic diary. If the participant uses nicotine, they will be trained to activate the 
electronic diary every time they use a nicotine product. Participants will wear the 
actigraphy wristwatch while they are awake and asleep. Before participants go to 
bed, they will be prompted to turn off the blood pressure device and set a wake-up 
alarm on the electronic diary for the following morning. Once they wake up from 
this alarm the following day, they will turn back on the ambulatory blood pressure 
monitor and put it on within 45 minutes. These 45 minutes allow for grooming, 
showering, and other morning routine activities. On two of the “full” monitoring 
days, participants will also wear the blood pressure monitor overnight, however 
they will not need to complete the hourly electronic diary questionnaires 
overnight.  
 
On the “partial” monitoring days, participants will not wear the blood pressure 
monitor or complete the hourly electronic diary interviews. The actigraphy 
wristwatch is worn on both “full” and “partial” monitoring days of the study 
(including every night of sleep during the monitoring phase).  
 
Several additional phone calls will be scheduled for the first or second day of the 
monitoring phase in order for a research assistant to answer any of the 
participants’ questions and asses any difficulties they may be having. Similarly, 
another phone call will be made on the third or fourth day of the monitoring phase 
in order to confirm that the equipment is still working correctly and to review 
instructions with the participant.  
 
On two separate non-“full” monitoring days during the daily monitoring period, 
participants will be instructed by a research assistant to complete the Automated 
Self-Administered Recall System (ASA24) outside the lab. 
 
Session 3: CARDIOVASCULAR HEALTH 
This visit will take place at Old Engineering Hall, will last (~3 hours), and will be 
conducted by a trained researcher. It involves returning the EMA equipment to 
research staff for data retrieval, assessment of pulse wave velocity and endothelial 
function, and additional questionnaires. 
 
EMA Device return: 
Participants will arrive to the visit wearing their devices. They will take off their 
devices and a research assistant will review the data that was collected over the 
10-day period. If it is determined that the participant did not complete the 
expected number of interviews and blood pressure readings, they will be asked to 
complete extra day(s) of monitoring to provide complete assessments.  
 
Participants will be asked about their compliance with pre-visit instructions (same 
as Visit 1 plus an additional instruction to wear loose clothing that allows research 
staff to attach heart-activity measuring devices without disrobing). Similar to the 
previous visits, these instructions will be given during a phone call made by a 
research assistant the day prior to the visit. While the research staff is reviewing 

 
 
 
 
 
18 
the participants’ daily monitoring data, participants will complete a number of 
psychosocial questionnaires.  
 
Cardiovascular Assessments: 
Following the return of the EMA devices, three separate (non-invasive) protocols 
will be administered to assess aspects of the participant’s cardiovascular 
health/function: 
 
Autonomic Function and Arterial Stiffness:  Self-adhesive  sensors will be applied 
to the participant (on the back of the neck, upper chest, shoulders, ribs, and calf 
area below the knee) to record their electrocardiogram and monitor changes in 
thoracic/calf impedance due to blood flow (dual-impedance cardiography). 
Continuous beat-by-beat BP will also be monitored using cuffs placed around the 
participant’s non-dominant arm and the index/middle fingers of the opposite 
hand. Recordings will be taken for 5 minutes while the participant is at rest and 
breathing normally, and also during a paced-breathing protocol (i.e., 11 breaths 
per minute). From these recordings we will gather key measures of HRV, BRS, 
and pulse wave velocity (PWV).  
 
Endothelial Health:  Venous occlusion plethysmography (VOP) will be used to 
assess the participants’ endothelial function. First, participants are comfortably 
seated and outfitted with the necessary equipment, including: an occlusion cuff 
for both their wrist and arm, and a strain gauge wrapped around their forearm. 
During a standard sequence of cuff inflations/deflations, the participants’ forearm 
blood flow is measured via changes in forearm circumference (as indexed by the 
gauge). Following “baseline” readings, the arm cuff is inflated to 40 mmHg above 
systolic pressure for 5 minutes until it is released and “max” readings re-
commence. This post-occlusion state is referred to as reactive hyperemia and 
provides a reliable index of endothelium dependent vasodilation. The participant’s 
max flow readings are compared to their baseline readings to derive a percentage 
increase, with greater values associated with better health. 
 
Pulmonary Function:  Participant’s lung function will be measured using 
spirometry. While standing, individuals are instructed to inhale/exhale into a 
mouthpiece under normal and forced/maximal conditions. From these tests a 
participant’s forced volume capacity and forced expiratory volume can be 
determined. 
 
Blood Draw: 
Lastly, a trained phlebotomist under the supervision of Dr. Anna Marsland (Co-I) 
will conduct a blood draw for inflammatory and metabolic measures and a hair 
collection sample to assess hair cortisol levels.  
 
Site specific laboratory safety policies when handling human blood samples will 
be followed including the use of Biological Safety Cabinets and Personal 

 
 
 
 
 
19 
Protective Equipment/PPE: gloves, lab coat/gown, glasses, and face mask at all 
times when working with specimens in the lab. 
 
Participants will be asked to abide by the following fasting restrictions prior to the 
session: nothing to eat or drink (including alcohol and caffeine) except water from 
9pm the night before; no exercise for 12 hours prior to the session; no 
nicotine/smoking 3 hours prior to the session; no cold/allergy or headache 
medication (aspirin, ibuprofen or other NSAID) for 12 hours prior to the session. 
A researcher will call the participant the night before the appointment and ask if 
they have taken antibiotics in the past 2 weeks, or if they have a current infection 
or cold symptoms (see attached Blood Draw Eligibility Questionnaire). If so, they 
will be rescheduled. Participants will also be rescheduled if they have had 
anything to eat or drink except water since 9pm the night before their appointment 
or report acute illness symptoms.   
 
Blood will be delivered to the Behavioral Immunology Laboratory (605 OEH) of 
Dr. Anna Marsland within 30 minutes of the time of the blood draw. Blood will 
also be delivered to UPMC Central Labs for glycemic and lipid assays.  
 
Hair strands: ~ 50 mg; 100-150 strands will also be collected with fine scissors as 
close as possible to the scalp from the upper part of the back of the head so that it 
is not noticeable. The samples will be retained by the Behavioral Immunology 
Lab and tested for cortisol levels.  
 
Session 4: MRI  
The fourth session will be an MRI Scan. It is anticipated that the complete session 
will take ~3 hours to complete, with less than 60 minutes in the scanner. 
Participants will be scheduled for the MRI scan and standard sequences will be 
collected that will consist of images of brain structure and function.   
 
This session will take place at the BRain Imaging Data Generation & Education 
(BRIDGE) MRI Center located at the Mellon Institute and will be conducted by a 
trained researcher. Once at the center, participants will have their seated BP and 
HR taken, and they will receive training for the MRI protocol that will be 
administered. An additional screening for MRI eligibility will take place as a 
safety precaution, in addition to a pregnancy test (if necessary). Participants will 
also be asked to remove any piercings and completely empty their pockets of all 
contents. They will be given ear protection against the sound of the MRI machine 
and asked if they need to use the restroom before beginning.  
 
Subjects will also be equipped with physiological monitoring (EKG sensors, a 
respiration belt, and a BP cuff) devices before being put into the scanner. The 
total time in the scanner will not exceed 60 minutes. 
 
MRI Protocol:  

 
 
 
 
 
20 
Structural MRI sequences:  Several structural sequences will be collected that will 
provide detailed information about brain morphology (T1 MPRAGE sequence) 
and hippocampal morphology (high-resolution hippocampal sequence). During 
these scans, participants will be asked to lay still and relax quietly.  
 
Functional MRI sequences:   Participants will be trained in all functional MRI 
scan tasks prior to entering the scanner. 
 
 
 
Participants will use a mirror and back-projection screen system with a response 
glove. Participants will be able to view a screen at the back of the scanner via a 
mirror fixed to the head coil. They will be given a response glove to wear in each 
hand with buttons located under each finger. Depending on the functional MRI 
task, participants will be instructed to press one of 4 buttons underneath the 
middle and index fingers of their left and right hands.  
 
Two functional MRI scans will involve identifying visual targets by pressing the 
correct button on a response box. For example, in one task, participants will see a 
series of color words (red, blue, yellow, green) and will be instructed to identify 
the color in which the word is presented as quickly as possible using response 
buttons on a key pad. In a second task, the participants will see numbers and will 
be asked to identify which one is different from the others. In the final task, 
subjects will view pictures of people and scenes. When viewing the pictures, they 
will be instructed to either allow themselves to experience any resulting emotions 
they feel about the picture naturally or to change how they feel by thinking 
differently about the picture. For example, when viewing a picture of a person in a 
hospital bed, then they can think of a quick and speedy recovery for that person. 
This is also called positive re-framing. As some subjects may find the nature of 
some of the pictures upsetting (e.g., they may feel disgusted if they see a picture 
of a dirty floor), we will allow them to view sample pictures before the MRI to 
prepare them. Only subjects who agree to view the pictures after practicing the 
task will be administered this paradigm during the scan. Participants completing 
the picture paradigm will also complete the Post-Task Questionnaire which 
assesses the strategies that were used to change their feelings about the pictures 
during the task.  
 
An additional functional scan will be taken while the participant is at rest. The 
participant will be instructed to relax and lie still while looking at a fixation cross 
on a screen while remaining awake. This scan provides information on the brain 
networks involved while an individual is at rest.  
 
Participants will be reminded periodically that they can discontinue the MRI 
protocol at any time. This will be done by a two-way communication system that 
allows for continuous monitoring and verbal exchanges with the participant 
throughout the MRI protocol. However, discontinuation of the MRI session may 
make them ineligible for continuation in the study. 
 

 
 
 
 
 
21 
Participants will be asked to provide subjective ratings of valence (1=very 
unhappy; 9=very happy), arousal (1=very calm; 9=very excited), and control 
(1=not at all; 9=very) during the MRI protocol. Participants will be familiarized 
with these rating scales beforehand. 
 
All participants will be given a squeeze ball to press in the event that they want to 
end the scan for any reason. An alarm will sound in the control room and the 
technician will stop the scan and remove the participant from the scanner 
immediately. 
 
1.5.4 POST RANDOMIZATION TASKS: 
Actigraph Link will be worn for one week every 6 weeks for each study 
participant. Measures of exercise self-efficacy are collected during the 3rd week of 
the intervention protocol. 
 
1.5.5 TIME POINT 2 (6 months): 
At 6-months into the exercise sessions, the participants will complete the 
assessments that were conducted at baseline. These assessments should be 
completed within a 1 month window; therefore 2 weeks before their midpoint date 
or 2 weeks after their midpoint date. The EMA measurement will be completed at 
month 5 to ensure all other key physiological assessments are completed on time.  
 
1.5.6 TIME POINT 3 (12 months – post intervention): 
At the 12-month session, participants will complete all the assessments that were 
done at baseline and 6 months. These assessments should be completed within a 
1-month window; therefore 2 weeks before their end date or 2 weeks after their 
end date. It is encouraged to complete session 1 and session 2 within 2 weeks of 
the intervention completion. If someone is going on vacation after the 
intervention, then they may be brought in during their last week of intervention 
for the 12-month assessments.  The EMA assessment will begin at Month 11 to 
ensure all other key physiological assessments are completed on time. 
 
1.6 
RANDOMIZATION:   
After the completion of the baseline measures and sessions described above the 
participants will be randomly assigned to one of two groups. These include (a) an 
exercise condition that will consist of 150 minutes per week of moderate-to-vigorous 
intensity exercise, or (b) a PA and health information group. We will use a computer 
randomization protocol that will incorporate a verification that all session visits have 
been completed and data entered into the database. We will use a minimization algorithm 
with equal allocation to one of the two groups.  The use of the minimization strategy will 
ensure treatment balance on the two factors of age at study entry (=<42, >42) and gender. 
Once randomized, an assigned staff member will contact the participant via phone with 
the group information. On this contact, the staff member will give information such as 
date, day, time, and location of the subject's first intervention session.   
 
1.7 
TARGETED ENROLLMENT TABLE  

 
 
 
 
 
22 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.8  
INTERVENTION: 
 
1.8.1  AEROBIC EXERCISE GROUP: Participants in the aerobic exercise 
condition will begin at a lighter intensity (50-60% Heart Rate Reserve or HRR) 
for 15-20 minutes during the first week of the program and gradually increase 
their intensity and duration by 5 minutes each subsequent week until they reach 
60 minutes of exercise depending on the group (see more below). All exercise 
sessions start and end with 5-10 minutes of stretching for the purpose of warming 
up and cooling down. Levels of exercise intensity will be prescribed based upon 
maximal responses during the initial graded exercise treadmill test and then 
reassessed after the midpoint graded exercise treadmill test. As the participants 
will be low active at baseline, the prescribed intensity will be 50–60% of the 
maximum heart rate reserve for weeks one to six and 75–85% for the remainder 
of the program as long as it is deemed safe by the monitoring exercise 
physiologist and trainer. Participants that feel comfortable increasing their levels 
at a more rapid pace will be allowed to do so if the accompanying trainer also 
agrees that it is safe. Participants in the aerobic group will wear a heart rate 
monitor and be encouraged to exercise within their target heart rate zone. Heart 
rate intensity will be measured by a Polar H10 heart rate strap that is worn below 
their chest. Certified exercise instructors will closely monitor attendance, 
intensity, frequency, and safety. On-site exercise sessions will occur two 
days/week for the duration of the intervention and at home for one other day of 
the week. Compliance to home-based exercise will be monitored by exercise 
diaries.  
 
1.8.2 PHYSICAL ACTIVITY & HEALTH INFORMATION GROUP:  The 
control group will be asked to wear an ActiGraph daily monitor for 7 days every 6 
weeks. They will not be given any information on exercise and they will not be 
asked to change their behavior or exercise patterns. 
 

 
 
 
 
 
23 
1.8.3 EXERCISE COMPLIANCE:  Participants in the study will be 
completing part of the exercise under supervised conditions and part of the 
exercise under unsupervised conditions (e.g., home exercise). Monitoring 
compliance during the supervised exercise is relatively straightforward because 
we will have trainers monitoring attendance, duration of exercise, perceived 
exertion, mode of activity, and heart rate intensity throughout the period. 
However, compliance during the unsupervised sessions (~30 minutes) is 
inherently more challenging. In this study, we have proposed a two-faceted 
approach for monitoring compliance during the unsupervised periods. First, 
within the first few weeks of the intervention, our staff will train participants on 
the Ratings of Perceived Exertion (RPE) Scale and participants will be instructed 
to keep weekly diaries of the times, durations, and RPE intensity during the 
unsupervised exercise that they engage in. These diaries will then be collected on 
a weekly basis and entered into our database (REDcap). These diaries are a 
natural component of training since training involves the identification of barriers 
and approaches to overcoming non-compliance while keeping individuals 
engaged and continuing to return for the sessions. 
 
In addition to the self-reported diaries of unsupervised exercise, we will have 
participants wear an Actigraph accelerometer for 1 week every 6 weeks of the 
study for 12 months (~9 weeks of accelerometer data). We will be able to use 
these data as objective confirmation that when participants are recording their 
exercise they are doing the activity at the prescribed intensity and for the 
prescribed duration. These assessments are important for providing an objective 
assessment of compliance during the unsupervised exercise periods. 
 
Based on the above, we have operationally defined compliance to the intervention 
as those participants meeting the prescription of an average of 150 minutes per 
week of moderate-to-vigorous intensity exercise.  Compliance to these 
prescriptions will be based on the information gathered from the weekly diaries, 
the supervised exercise sessions, and the accelerometer recordings. We will 
record on a weekly basis for each participant whether they were compliant or not.  
 
Our analysis plan has proposed an intent-to-treat analysis where all participants, 
regardless of whether they complete all sessions, will be invited to return for 
follow-up assessments. However, there will likely be some participants that refuse 
to return for follow-up assessments or miss a mid-point assessment. “Lost to 
follow up” will be defined here as missing data that is contributing to the primary 
outcomes. The reason will be recorded (i.e., missed visit due to traveling, illness). 
These criteria and documentation will also be applied for any discontinued 
subjects with the reasons for discontinuation being recorded (i.e., moving out of 
state).   
 
Regarding the analytical approach, we propose to use all available data in the 
planned analysis for testing our primary outcome. Since we are examining 
changes over time we will be able to conduct the analysis as long as there is at 

 
 
 
 
 
24 
least one follow-up assessment (either at the 6-month time point or the 12-month 
time point). We will assess the missingness of data consistent with the 
assumptions of the modeling approach described above. That is, we will examine 
the randomness of missing data and determine if the data is missing completely at 
random (MCAR) or missing at random (MAR) and use maximum likelihood 
estimation for computation of unbiased parameters estimates and standard errors. 
We will not conduct multiple imputation to correct for missing data. 
 
 
1.9 
PARTICIPANT SAFETY: 
The safety of study participants will be monitored throughout the study. Study 
participants will undergo an extensive screening prior to entering the trial to determine 
that it is safe for them to participate in the intervention. Safety is continuously monitored 
during the intervention via two supervised sessions per week. We are aware of the 
potential for serious adverse events to occur with any type of exercise training. As such, 
the trial will take measures to reduce the risk of an adverse event. All individuals 
involved in human subject’s research, regardless of whether exercise training is involved, 
are trained and certified in CPR and First Aid. We will have a medical monitor 
(cardiologist) who will review any exercise ECGs that are flagged for any unusual 
reading. Included in the exercise stress testing room is an automated external defibrillator 
(AED), should it be deemed necessary. Lastly, protocols are currently in place to respond 
to any adverse events that include contacting emergency response personnel and 
facilitating their arrival to the correct rooms.  
The Data Safety Monitoring Board (DSMB), is responsible for monitoring study data for 
evidence of adverse effects attributed to the intervention. The DSMB also has the 
authority to recommend changes, stopping the trial at any time, or pausing the trial until 
problems are resolved. All AEs experienced by the participants during the study (Consent 
signing until end of study outcome collection) are to be reported during the regularly held 
DSMB meeting. The eBACH study will track the occurrence of: 
•  Serious Adverse Events (SAE) 
•  Unexpected Events  
•  Unfavorable medical events that occur during intervention 
sessions 
 
The trial has created two forms for adverse event reporting. These documents have been 
created based on the belief that trainers and staff should not be responsible for 
determining whether the event is related to the intervention or whether/how it should be 
reported to the IRB and DSMB. Only the PI will determine if the event is (a) expected or 
unexpected, and (b) related to the intervention. Given the frequency of the interactions 
between subjects and trainers it will be important for trainers and staff to ask if the 
participant has had any illnesses, injuries, or medication changes since their last 
supervised exercise session. Based on responses to these questions, regardless of 
seriousness or relatedness, the trainer will complete a form documenting an adverse 
event. We have created a brief form for trainers (eBACH Adverse Event Form for 

 
 
 
 
 
25 
Trainers) for this purpose. Trainers or staff would first document the event on this form. 
This form allows trainers to circle the reported event (or provide an event not listed on 
the form). This completed document would be sent to the project coordinator who would 
then speak to the trainer and/or participant to find out more information about the event. 
The project coordinator would then complete the eBACH Event Evaluation Form based 
on this communication. This form has several sections with boxes available for 
documenting the reported severity (mild, moderate, severe), the chronicity (single 
occurrence, intermittent, persistent), whether the event was resolved or not, and whether 
it was related to the intervention and expected or unexpected. In particular, these forms 
are intended to clarify for the investigators, the IRB, and the DSMB, whether the adverse 
event was serious, whether the adverse event was related to the intervention, and whether 
it was expected or unexpected. An example of an expected adverse event from the 
exercise intervention would be muscle soreness which would be listed as a known risk in 
the IRB. An unexpected adverse event related to the intervention would be if someone 
falls off a treadmill and injures an ankle. The PI will oversee the determination of these 
events and if there is any question about whether the event was related to the 
intervention, its seriousness or expectedness, the event will be discussed at the weekly 
project meeting. For DSMB purposes, all adverse events that are reported will be 
recorded and separated based on whether the event was related to the intervention, 
unexpected, its seriousness, severity, chronicity, and resolution. 
 
1.10 
DATA MANAGEMENT: 
We appreciate the challenges associated with the organization, execution, management, 
and analysis of a study of this kind and recognize the importance of frequent 
communication between staff, students, analysts, and investigators. Protection of subject 
privacy and safety, meticulous data quality checks, and prudent organization is the 
bedrock for success of a study like this, which will require a sizeable team/staff.  
IT staff will control permissions to the database to ensure proper access and data 
management by staff, students, and investigators. We will store and link all behavioral 
and assay data on a HIPPA secure cloud-based server (REDcap).  
The data forms in REDcap are similar to the actual paper version of the forms that will be 
completed by participants and staff. All participant data will be stored and archived on a 
secure server at the University of Pittsburgh. The server is set for safe mode that allows 
two copies of each data to be saved on the server. By having the data base on REDCap, 
all data is secure is case of a natural disaster in which the server would be completely 
undamaged.   
 
Missed Visits: All outcome collection must be reported and stored in the database.  
Therefore, if an outcome visit is missed or not collected, a missed visit form must be 
completed in REDCap. Once a missed visit form is completed, the data management 
team will indicate in the database that the outcome data for the specific measure is missed 
to follow up. All missing data in the database will require additional documentation 
regarding whether the data are “lost to follow up” or have been collected but there was a 
problem with it. 

 
 
 
 
 
26 
 
The missed visit form has three sections that must be completed: 
• Time-point of the missed visit 
• Outcome that was missed 
• Reason the outcome was missed 
 
If a study participant misses multiple outcome measures during the same time point, one 
missed visit form can be completed if the reason missed is the same for each measure. 
Thus, staff do not have to complete a new form for each missed outcome for the same 
time point. If this study participant would also miss post intervention visits, a new form 
must be generated since this is a different time point. 
 
1.11 
QUALITY CONTROL: 
There will be multiple layers to the quality control process. First, we will have double 
entry for all paper-pencil assessment measures that need entered into REDCap to ensure 
that data is entered correctly. A separate person will review the data for any 
discrepancies. Before randomization will occur, the study coordinator will check all data 
for completeness and quality. At which point, the study coordinator will complete the 
data management quality checklist and give approval for randomization to occur. At 
weekly meetings the data will be reviewed to identify any issues with QC.  
1.11.1 MRI QUALITY CONTROL: 
Research associates (RAs) trained in MRI data acquisition and administration of 
tasks as well as placement of physiological equipment (e.g., blood pressure cuff) 
will be present at every scan. These RAs will be accompanied by another research 
staff member to assist in participant instruction and monitoring of physiological 
data during the scan. A trained MRI technician from the BRIDGE Center will be 
present to operate the MRI scanner and Siemens software, orienting all field of 
view maps and ensuring proper slice placement. Data acquired in the MRI 
scanner will be distributed to the Erickson and Gianaros labs in parallel for 
simultaneous quality checks and maintenance of copies of original data. Data will 
enter a quality check pipeline that includes 4 quality control steps: (a) checking of 
correct image acquisition parameters, (b) an assessment of image quality 
parameters (i.e., signal-to-noise, contrast-to-noise, etc.), (c) an evaluation of 
appropriate slice orientation and brain coverage for each sequence, and (d) 
preprocessing to ensure minimal movement in fMRI scans. 
1.11.2 ACTIGRAPHY, FITNESS, EMA, CARDIOVASCULAR QUALITY 
CONTROL:  
All actigraphy, fitness, EMA, cardiovascular assessments, and questionnaires will 
be interrogated at baseline before randomization to confirm data quality 
(including means, standard deviations, out of range values, etc.).  Quality control 
assessments will continue on a bi-weekly basis and values will be reported to the 
Principal Investigator for possible discussion during weekly project meetings.   

 
 
 
 
 
27 
1.12 
RESOURCE SHARING: 
1.12.1 DISSEMINATION PLAN:   
First, the trial will be registered at ClinicalTrials.gov after formal approval of the 
protocol by the Data Safety and Monitoring Board, which is expected to be 
confirmed within the first 6 months of funding. Once the protocol is approved by 
ClinicalTrials.gov, updates to recruitment and status of the project will be 
provided to NHLBI every 3 months, or more frequently if requested, until the 
closure of the study. Finally, results from the primary and secondary aims will be 
uploaded to ClinicalTrials.gov within 12 months of the completion of the clinical 
trial. Dr. Erickson has significant experience working with ClinicalTrials.gov in 
other clinical trials and will be responsible for seeing that these results are 
updated in a timely fashion. 
 
In addition, participants will be informed of the posting of information to 
ClinicalTrials.gov as part of the Informed Consent process and will be informed 
that there will not be any individual data uploaded or any private or confidential 
information shared on the ClinicalTrials.gov website.  
 
Finally, the University of Pittsburgh has internal policies in collaboration with the 
Human Research Protection Office (HRPO) to ensure that the registration and 
result reporting are in compliance with federal policy. Dr. Erickson will work 
with the Institutional Review Board, the Data Safety and Monitoring Board, and 
the University HRPO to ensure that these policies are followed.   
 
We will develop an extensive dissemination plan and will publicize the results of 
the study to practicing clinicians, policy makers, research study participants and 
the general public by utilizing diverse strategies outlined below. Dissemination 
will occur through the following techniques: (a) media coverage through press 
releases and interviews targeted to local and national newspapers, television and 
radio outlets; (b) production of a research summary document and “facts sheet” 
targeted to the general public which clearly and concisely summaries the key 
conclusions of the trial; (c) production of flyers, posters, brochures, and research 
briefs targeted to broad audiences; (d) study newsletters targeted to study 
participants; (e) utilizing our study website to share news and research findings to 
study participants and the public; (f) publishing the results from the trial in high 
impact and high quality scientific journals within 12 months of closure of the 
study. 
 
The trial has created a publications policy structure that also provides guidelines 
to students and investigators about authorship, data analysis and accessibility 
approaches, etc. 
 
1.12.2 DATA SHARING: 
We are familiar with the NIH policies regarding data sharing and will comply 
with local, state, and federal laws, such as the Privacy Rule, a Federal regulation 
under the Health Insurance Portability and Accountability Act (HIPAA). We will 

 
 
 
 
 
28 
follow the NIH data sharing guide and provide a data sharing plan to be reviewed 
and approved by the relevant NIA PO. Data sharing will be accomplished using 
mixed modes, each providing a different level of access. This will include data 
enclave (controlled, secure environment in which eligible researchers can perform 
analyses using data resources), researcher’s efforts (investigator responds to data 
requests), and publishing articles in scientific publications. Each of these methods 
will require investigators to complete data request forms to document aims, 
procedures, and to ensure that all investigators understand the policies put into 
place by the study team.  We propose to use REDcap (Research Electronic Data 
Capture) as a data enclave for investigators that may request access to certain 
data. REDcap is a secure web application for building and managing surveys and 
databases.  It allows a streamlined process for rapidly creating and organizing 
databases and allows for automated exporting of data to Excel and common 
statistical packages (SPSS, SAS, Stata, R) as well as a built-in project calendar, a 
scheduling module, ad hoc reporting tools, and other features.  
 
1.12.3 DATA SHARING AGREEMENT: 
The data-sharing agreement provides for: (1) a commitment to using the data only 
for research purposes and not to identify any individual human participant; (2) a 
commitment to securing the data using appropriate computer technology; (3) a 
commitment to destroying or returning the data after analyses are completed, (4) a 
commitment to focus on particular aims documented in the agreement, (5) a 
commitment to work with the Principal Investigator of the project to ensure 
accuracy of analyses and interpretations, and avoid errors. We will make the data 
available to outside investigators using a data sharing agreement after publication 
of the primary aims and at completion of quality control assessments and data 
organization. Protecting the rights and privacy of human subjects will be our first 
priority. The final datasets will be de-identified prior to release for sharing. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
29 
1.13 
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Colcombe, S. J. et al. Aerobic exercise training increases brain volume in aging 
humans. The journals of gerontology. Series A, Biological sciences and medical 
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Voss, M. W. et al. Plasticity of brain networks in a randomized intervention trial 
of exercise training in older adults. Frontiers in aging neuroscience 2, 
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Chaddock, L. et al. A neuroimaging investigation of the association between 
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Gray Matter in the Cardiovascular Health Study. Journal of Alzheimer's disease : 
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Voss, M. W. et al. Neurobiological markers of exercise-related brain plasticity in 
older adults. Brain, behavior, and immunity 28, 90-99, 
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Burdette, J. H. et al. Using network science to evaluate exercise-associated brain 
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32 
 
 
 
 
 
 
 
 
 
Amendments in Study Protocol 
 
 

 
 
 
 
 
33 
Given the continued challenges of navigating the COVID-19 pandemic, we requested 
to modify our recruitment/randomization accrual and targeted sample size for the study 
titled “Exercise on Brain and Cardiovascular Health (eBACH)”, Project 3 of P01-
HL040962, on September 28th, 2021.  These recommended changes were approved by 
the meeting of the Data Safety and Monitoring Board on September 2nd, 2021. The 
details of this requested change are described below. 
 
• Our original targeted sample size of 150 was re-evaluated by our study statistician 
(Dr. Chaeryon Kang, Department of Biostatistics, University of Pittsburgh) with a 
focus on the minimal sample size needed to be sufficiently powered to test our 
primary hypotheses. Based on prior literature described in the grant application, we 
estimated effect sizes between .5 and .6 (Cohen’s d) for changes in brain volume 
resulting from an exercise intervention. Using mixed effects modeling of the data in 
an intent-to-treat approach, we will have between 68-83% power to test our primary 
aim (hippocampal volume change) with 120 participants, which accounts for a 
possible 20% attrition rate. Similar effect sizes for the effects of exercise on various 
cardiovascular disease biomarkers have been reported (between .58 and .6). 
Therefore, a sample of 120 would provide us >80% power even with 20% attrition 
to test our hypotheses about the effects of exercise on cardiovascular disease 
biomarkers and mediators. In addition, we will be sufficiently powered (>80%) to 
test several of our secondary aims (e.g., depressive symptoms and functional brain 
connectivity) with a targeted sample of 120. In sum, we have determined that we 
will have sufficient power to test our primary aims with a reduction in the targeted 
sample size to 120. What is more at risk with a reduction in sample size is the 
statistical power for mediation analyses to test our tertiary aims (i.e., to use 
structural equation modeling approaches for examining mediators between changes 
in brain and changes in cardiovascular health markers). As such, we propose to 
move forward by conducting these tertiary aims on an exploratory basis. 
• The power estimations described above include an attrition rate of 20%, but our 
current attrition rate is <10% for all participants that were not disrupted due to 
COVID. This suggests that the power estimations described above are likely 
conservative estimates. 
• Our current exercise adherence and compliance rates are >90% indicating that we 
are successful at getting and keeping participants physically active. This is important 
because low adherence and compliance have a direct effect on our ability to test our 
hypotheses (i.e., if participants are not 
• adherent then we would be unable to test our aims). In short, the exercise sessions 
and intervention are going well which supports our ability to test our primary and 
secondary aims with a smaller than originally proposed sample. 
• The modified accrual rates described in our letter in December 2020 were overly 
optimistic at the time and proposed in view of the forthcoming public availability of 
vaccines and potential normalization of research conduct prior to the rise of SARS-
CoV-2 variants. Unfortunately, the year 2021 in the Pittsburgh region has been 
marred by unanticipated challenges including variable vaccine hesitancy rates 
across different districts, the rise of the delta and other variants, continued illnesses 

 
 
 
 
 
34 
and hospitalizations, need for quarantine, reduced density of staff and participants in 
buildings, school and day care cancellations, etc. (see more on this below). As such, 
we propose to modify our accrual rates by taking advantage of a 1 year no cost 
extension (which was not included in our adjusted targets in December of 2020). 
We have worked closely with our budget office to confirm that we will have 
sufficient funds for a no cost extension and reducing our proposed targeted sample 
from 150 to 120 (30 participants or 60 sessions) will also reduce additional costs 
and time. This new accrual rate is more feasible given current trends in the COVID 
pandemic. 
• There are two primary bottlenecks that we have experienced related to our lower-
than-expected accrual rate. First, our study team has experienced recent turnover in 
staff that were hired for recruitment and screening. This was noted by the study PI 
and DSMB members at the September 2nd, 2021 meeting. To address this issue, we 
have submitted hiring materials to the University of Pittsburgh Human Resources 
office to hire a new staff member who will be dedicated to advertising, screening, 
and recruitment for the study. We expect this individual to start in October 2021. In 
the interim, we will immediately have staff from other Projects and Cores assist in 
recruitment and screening efforts. Secondly, we have had an unprecedented number 
of session cancellations and participants withdrawing from the study during 
baseline assessment sessions (before randomization). As such, our screening-to-
randomization ratio is lower than anticipated which requires the study team to 
screen more individuals to randomize one person to the intervention. This behavior 
(session cancellations and withdrawing before randomization) has been observed 
across several ongoing studies and can be attributed to issues related to the 
pandemic described above (e.g., need for quarantine). We are currently working 
through methods to accommodate missed sessions or make participation in the trial 
more flexible and accommodating to the needs of the participants. 
• The DSMB members noted on September 2nd, 2021 that the rate of minority 
recruitment is lower than anticipated. We are taking active steps to improve this rate 
including community advertisements in addition to our marketing strategy that we 
have used up until now. Dr. Gianaros has also been attending local farmer’s markets 
to recruit within diverse neighborhoods in Pittsburgh, and we have a forthcoming 
meeting with an elected Councilperson in the Wilkinsburg district of Pittsburgh to 
formulate a community-based recruitment effort to reach out to a more diverse 
sample to meet enrollment targets. Once our new staff are hired and trained, we will 
be actively implementing these strategies to enhance minority recruitment. If 
necessary, we will pause the recruitment of non-Hispanic whites into the trial in 
order to focus on increasing enrollment of underrepresented minority groups. 
• Lastly, as noted in the attached and updated accrual plan approved by the DSMB, 
we can report that we are currently 4 people lower than our randomization target as 
of this writing. Hence, 57 of 61 people have been randomized, which reflects 93% 
of the new DSMB approved randomization goal for this point in time.