The Effect of Physical Activity on Cognition Relative to APOE Genotype (PAAD-2)
Phase: NA
Conditions: Healthy, Dementia, Alzheimer Disease, Physical Activity
Sponsor: University of North Carolina, Greensboro

Study Protocol and Statistical Analysis Plan 
Study Title:  The Effect of Physical Activity on Cognition Relative to APOE Genotype (PAAD-2) 
Date:  May 24, 2021 
NCT:  ID not yet assigned 
 
 
 

Taken from the Specific Aims Section:  The purpose of this study is to test the following specific 
aims. 
• 
Aim 1: Test the causal link between PA and cognitive performance in persons with a family 
history of Alzheimer’s disease (FH+) 
• 
Aim 2: Determine if the effect of PA on cognitive performance is moderated by APOE4 
status. 
• 
Aim 3: Assess the extent to which the effects of PA on cognition are explained by a 
moderated mediation model with cerebral structure, white matter integrity, and resting state 
connectivity as mediators and APOE4 status as a moderator. 
• 
Aim 4: Assess putative biomarkers as mediators and APOE4 status as a moderator of the 
effects of PA on cognitive performance. 
 
3.c APPROACH 
Our methods are based on our successful Phase I proof-of-concept trial (PAAD) in which we 
demonstrated cognitive benefits in persons with FH+ in association with 8 months of PA [1]. To logically 
extend that work, we will conduct a 12-month RCT, include MRI and more biomarkers, and incorporate 
further cognitive measures for comparison with findings from IGNITE. Participants will be randomly 
assigned to a PA condition (PAC) or a usual care control (UCC). Participants will be tested at 3 time 
points (pre, mid, and post) relative to the treatment period. We will use latent growth curve modeling to 
test questions related to trajectories of change.  
3c.1 Overview 
We will recruit 240 sedentary, middle-aged (40-65 years), cognitively normal adults with a FH+ to 
participate. Research staff will initially assess eligibility during a telephone interview. Prior to beginning 
the program, individuals will complete additional screening and sign an informed consent form at the 
pre-test. Those who remain eligible will be randomly assigned to the PAC or the UCC. Testing will take 
place at 0, 6, and 12 months. Following program completion, PAC participants will be encouraged to 
continue with their exercise independently, and UCC participants will be given a short-term YMCA 
membership or cash equivalent.  
3c.2 Participants  
3c.2.1 Recruitment. Cognitively normal, community-dwelling men and women 40-65 years of age 
will be recruited from six counties in North Carolina: rural (Randolph, Rockingham), regional city and 
suburban (Davidson, Alamance), and urban (Guilford, Forsyth) and other counties within a 2-hour 
distance of Greensboro, NC. This recruitment strategy will increase the diversity of our sample and 
contribute to the reproducibility of the results. The 6 counties have a combined population of 
approximately 469,588 in this age range [2]. We will advertise the study via local television, radio, social 
media, and newspapers and flyers distributed to support groups (see Letters of Support). 
Advertisements will request participation by adults (40-65 years) with a FH+ (defined as one first-
degree relative diagnosed with AD). Although APOE4+ make up approximately 24% of the general U.S. 
population <65 years of age [3], based upon our own [1, 4] and other research [5], recruiting adults with 
a FH+ will increase the percent of APOE4+ to ~35%. Thus, by recruiting 240 people with a FH+, we 
anticipate successfully enrolling about 80 APOE4+ participants. Given demographics of the recruitment 
region, we will include approximately 125 women and 115 men. In our past work, we have successfully 
recruited, screened, and enrolled participants from a similar age group (50-65 years) using these 
methods. We successfully enrolled 54 participants in Guilford County alone (target population=76,000) 
in a shorter period of time and with less funds allocated for recruitment. Assuming a similar success 
rate (0.07%) and given the size of the target population in the 6 counties (N=469,588), we will readily 
meet our target sample size (0.07%*N=328). We have letters of support from all of the YMCAs 
indicating their willingness to work with us in the enrollment of participants and the implementation of 
the program. 
  3c.2.2 Inclusion criteria. Inclusion criteria will be assessed during the telephone interview. 
Eligible participants must have a self-reported FH+ and be between 40-65 years of age, able to 

communicate in English, and sedentary (30 min of moderate intensity PA fewer than 3x/week for the 
last 3 months by self-report) as defined by the Guidelines of the American College of Sports Medicine 
(ACSM) [6]. We will not include currently active adults because they are unlikely to benefit further from 
the intervention, and are likely to exhibit ceiling-level cognitive performance that could bias statistical 
models. Previously, when recruiting people who are “not regularly physically active”, only 6% were 
excluded because of being too active [1]. 
3c.2.3 Exclusion criteria. Exclusion criteria will be assessed during the telephone screening, 
through surveys, and during pre-testing.  Decisions regarding inclusion/exclusion will be made by the 
Project Coordinator and the Cognitive Testing Post-Doc in consultation with the PI and Dr. Tomika 
Williams (Adult Gerontological Primary Care Nurse Practitioner). See Figure 2 for percentages 
excluded based upon these criteria in our previous study [1]. Individuals will be excluded if they: 
o Meet the criteria for clinical cognitive impairment (MCI, AD, or other 
forms of dementia) assessed in two stages. First, during telephone 
screening, we will use the modified Telephone Interview for 
Cognitive Status (TICS-m). The TICS-m has acceptable sensitivity 
and specificity in the detection of dementia [7] and amnestic MCI [8] 
and does not have the same ceiling constraints as other measures 
of cognitive impairment [8, 9]. Participants who score <36 will be 
excluded [8]. Second, at the pre-test, participants will complete the 
Montreal Cognitive Assessment (MoCA). We include people scoring 
equal or higher than 25 and exclude people scoring equal or lower 
than 22 without additional consideration. For people scoring 23 or 
24, the MoCA-MIS cutoff (< 11) is additionally considered to screen 
out individuals with potential mild cognitive impairment (MCI). For 
people meeting both MoCA-TS (≤ 22) and MoCA-MIS (≤ 10) may be close to MCI rather than 
normal cognition based on Kaur, Edland, and Peavy (2018)’s data [10]. These stratified and double-
layered criteria will maximize true positives and minimize false positives for the detection of MCI as 
distinct from normal cognition. Participants excluded based upon these criteria will be referred to Dr. 
Williams and advised to discuss their cognitive performance with their physician. Previously, 9% of 
interested participants (50-65 years) were excluded for this reason (see Figure 2). We anticipate 
fewer will be excluded in this study because of the expanded age range (40-65 years). 
o Cannot perform PA because of known cardiovascular, metabolic, or renal disease and are 
symptomatic (assessed using the 2014 PAR-Q+) or because of orthopedic limitations as per the 
ACSM Guidelines [11]. 
o Self-report any history of confounding neurologic (e.g., traumatic brain injury, prior stroke, 
myelopathy, myopathy, peripheral neuropathy, brain tumors), psychiatric (e.g., active major 
depression, any history of schizophrenia or bipolar disorder), or active severe or functionally 
disabling neurologic or medical diseases (e.g., Parkinson’s disease, active treatment for cancer), or 
any other conditions that might limit exercise or pose a danger to the patient – assessed using a 
Medical Health History (MHH). 
o Report the current use of medications to treat symptoms of AD or that adversely affect cognition 
(MHH). 
o Meet the criteria for depression (score >16 and anhedonia or dysphoria nearly every day for the 
past 2 weeks, plus additional symptoms in 2 or more other DSM symptom groups reported as 
occurring nearly every day for the past 2 weeks or 5-7 days in the past week) using the short form 
of the Center for Epidemiological Studies Depression Scale Revised (CESD-R). Report suicidal 
ideation on the CESD-R.  
o Are not able to be compliant with the study protocol (i.e., are unwilling to be assigned to either 
condition, will not live in the area through the completion of testing, are unwilling to complete all 
testing, are unable to attend the exercise sessions) 
3c.3 Procedures 

3c.3.1 Telephone interview. Interested individuals will be asked to contact project staff by 
telephone. Staff will describe the study procedures. Individuals who remain interested will answer 
questions relative to the inclusion and exclusion criteria and will complete the TICS-m to ascertain initial 
eligibility for the study. Eligible participants will be scheduled for pre-testing. Following the telephone 
interview, participants will be contacted by the Cognitive Testing post-doctoral fellow to complete 
additional surveys (e.g., the Medical Health History [MHH], the CHAMPS, demographics).  This will 
provide additional contact with participants between telephone screening and pre-test which should 
reduce dropout, will reduce participant burden at the pre-test by reducing the time requirement, will help 
establish a rapport with the staff, and will allow participants more time to obtain a physician consent 
before the pre-test.  Upon review of the MHH, participants will be identified as: (1) apparently healthy, 
(2) having known disease but asymptomatic or with managed symptoms, or (3) symptomatic. 
Participants in category 3 who have known disease that is poorly managed (i.e., they are symptomatic 
and unstable) will not be allowed to participate. Other participants in category 3 and participants in 
category 2 must provide physician consent prior to participation.  Those in category 1 will be asked to 
obtain physician consent for participation. 
3c.3.2 Pre-, mid-, and post-test. At the pre-test, participants will read and sign an informed 
consent approved by the Institutional Review Board, will complete measures regarding exclusion 
criteria (see 3c.2.3), will complete the MoCA, and will provide a saliva sample for APOE genotyping.  At 
the pre-test, mid-test, and post-test, participants will come to the lab for two days of testing.  On the first 
day, participants will perform cognitive tests (see 3c.4.3), have resting heart rate (HR) assessed, and 
complete a submaximal exercise test (see 3c.4.4). On the second day, participants will provide a blood 
sample and complete an MRI scan (see 3c.4.5). On this day, testing will begin before 9:00 am to 
control for diurnal variability and with participants in a fasted state. Participants will provide a blood 
sample and then be offered a light breakfast. Participants will then complete an MRI scan (see 3c.4.5).  
3c.3.3 Experimental conditions.  Following the pre-test, participants will be randomly assigned to 
treatment conditions. Random assignment will be conducted using a computerized randomization 
procedure implemented by Labban (Co-I); participants will be contacted and informed of their group 
assignment by the Project Coordinator. Staff conducting testing (post-doctoral fellows, graduate 
research assistants) will be blinded to group assignment throughout the study.  Participants will be 
instructed not to discuss group assignment during testing sessions. These steps will contribute to the 
scientific rigor of the proposed study. 
3c.3.3a Physical activity condition (PAC). The PAC was used in our previous research in 
which we found improvements in memory in association with the program. It was originally based on 
meta-analytic evidence [26] indicating that in RCTs the largest effects of PA on cognition in older adults 
were in programs that include both aerobic and strength training (g=0.59). Hence, the PAC will include 
both modes of activity. Subjects will be asked to attend 3x/week for 1 year and exercise will take place 
in a group setting. Each subject will be encouraged to walk at a moderate intensity (target HR = 40-
59% HR reserve) dependent on resting heart rate and age [168]. At every session, exercise specialists 
will record the amount of time spent in the aerobic and strength training portions of the program, HR 
(assessed by palpation for 20-seconds) and RPE from each participant during the middle portion of 
aerobic activity, and RPE during the middle portion of strength training. Participants will record the 
number of sets and reps and the color of the exercise band or the weight (if/when individuals progress 
to handheld weights) used for each exercise during strength training.  
Every 3 weeks, Karper will observe a session to confirm that prescribed elements are included 
and non-prescribed elements have not been introduced. At this time, data from exercise logs and 
exercise specialist records will be reviewed for evidence of progression, consistent attainment of 
moderate intensity, and with respect to the prescribed duration of the aerobic and strength training 
components.  Karper will also assess resting heart rate (HR) and average HR during exercise using 
Polar HR monitors. This information will be examined and used to recalculate target HR. Action values 
are operationalized as moderate intensity exercise (RPE = 12-13, HR = 40%-59% HR reserve) with 
time in aerobic exercise increasing from 15-min/day in week 1 to 30 min/day by week 4 and with time in 
strength training at a minimum of 30/min day throughout. In the first weeks of the PAC, more time will 

be needed to teach the participants the exercises and to identify appropriate resistance levels.  
However, by week 8, strength training will be completed in 30 min; this will be maintained throughout 
the 12-month intervention. For strength training, elastic Therabands and dumbbells will be used. 
Subjects will begin with Therabands with the least resistance, completing one set of 10 repetitions for 
each of 10-15 exercises. As they can complete 15 repetitions for any given exercise in proper form, 
they will be progressed to the next higher resistance band for that exercise. If remediation is necessary, 
Karper will work with the exercise specialist to identify barriers and facilitators to treatment fidelity and 
will develop solutions to identified problems.  
This PA program is inexpensive, safe, and suitable for community adult programs and home-
based exercise recommendations. The program was initially implemented at local YMCAs but has been 
offered via Zoom since resuming post-COVID-19 pandemic. Fidelity across sites will be ensured by 
using qualified and experienced Exercise Specialists who will be trained by Karper (Co-I) to implement 
the program. Fidelity to the program and consistency of implementation across sites will be further 
ensured by Karper’s regular (once / 3 weeks) visits to each site to observe, evaluate, and provide 
feedback on sessions.  Measures of compliance and adherence will be obtained at all exercise 
sessions; sites will be compared monthly to ensure no substantive differences across sites. If such 
differences occur, we will determine their causes and make necessary changes. 
The exercise specialists will have an appropriate educational background, exercise leadership 
experience, ACSM Group Exercise Instructor Certification (or equivalent), and CPR Certification. All 
exercise specialists will be vetted by Karper in consultation with the PI (Etnier), will be provided up to 4 
hours of training by Karper to administer the program, and will be observed every 3 weeks. As 
described, we also have a well-thought out plan to ensure the fidelity of the intervention delivery. 
3c.3.3b Usual-care control (UCC). We will use a UCC in which we ask participants to maintain 
their normal health practices (e.g., diet, annual physicals) for 1 year. Because the inclusion criteria 
require that participants be sedentary, we anticipate that these individuals will not show consistent 
increases in PA over the year. Importantly, we considered the use of a comparison condition that 
controls for aspects of our intervention not specifically tied to the exercise itself (e.g., social interactions, 
attention, feelings of accomplishment). For the two conditions to be equal in all ways except the 
physical activity, this comparison condition would have to be engaging enough to ensure adherence for 
3 days/week for one year.  Thus, we considered non-exercise conditions such as health education, 
music lessons, and art lessons. However, interest in participating in these activities for an entire year 
might be very individualized; participants might not be willing to be randomly assigned to either group 
and might have low adherence if assigned to an “undesirable” condition. Further, these activities might 
themselves prompt participants to become more physically active and/or lead to cognitive gains, 
thereby reducing the expected treatment effect [12]. Alternatively, we also considered an exercise 
condition with less intense physical activities, such as a stretching and toning condition (STC). But 
given the evidence that an STC results in cognitive benefits indistinguishable from those in an exercise 
condition [13, 14], we determined an STC might limit our ability to observe treatment effects. Thus, we 
elected to use a UCC. To reduce effects from experimenter attention, to minimize attrition, and to 
assess possible cross-contamination, we will contact them once per week.  We will provide educational 
materials (covering health topics, but not PA) to UCC participants biweekly.  We will also contact UCC 
participants once per month to inquire about their health and to entertain questions about the 
educational materials.  Once per month, we will assess their self-reported PA [15]. In this fashion, UCC 
participants will be contacted by staff every week. To further encourage retention, we will provide UCC 
participants with a short-term YMCA membership or cash equivalent after the post-test. When 
participants are offered an intervention after a UCC waiting period, cross-contamination is low (7.1% of 
studies) and fewer participants drop out from a UCC than an exercise treatment (4.7% fewer) in trials 
up to 1 year [15].  
3c.3.4 Adherence. In our past research (PAAD), we anticipated 15% dropout; actual dropout was 
13% before program completion (all for reasons unrelated to the study) [1]. Completers attended 76% 
of prescribed sessions. We will incorporate the same methods to ensure high levels of adherence: 1) 
use a group-based program; 2) offer incentives for adherence; and 3) provide compensation for testing 

sessions. If participants drop out prior to pretest, we will recruit more participants to achieve our target 
sample size. Overall, we anticipate that 204 of the 240 participants enrolled will complete the study.  
3c.4 Measures 
3c.4.1 Genotype. Saliva samples will be collected using Oragene-500 kits.  Genomic DNA will be 
extracted from saliva samples for SNP testing. The SNPs associated with the two amino acid residues 
(codons 112 and 158) will be used to identify participants as APOE+ or APOE-. Remaining DNA 
material will be stored indefinitely for future analyses. 
3c.4.2 Biomarkers. We will use standard protocols for collection and storage of blood samples and 
assays and analyses. Blood samples will be collected from an arm vein at rest [16]. Plasma and serum 
will be separated by centrifugation. Samples will be stored at -800.  Glucose will be analyzed using a 
commercially available assay kit and requires <10 µL of serum. All other assays will be conducted 
using a multiplex system (Luminex 200S), which uses very small (20-50 µL total) volumes of blood. 
BDNF will be given priority in any insufficient samples. Samples will be stored indefinitely. 
 3c.4.3 Cognitive Test Battery. We will assess performance across cognitive domains, specifically 
including tests assessing cognitive abilities sensitive to the early stages of dementia [17] and to PA 
interventions [18]. We will include measures from our previous PAAD study [1] as follows:  information 
processing speed will be assessed with the Wechsler Adult Intelligence Scale (WAIS-IV) Digit Symbol 
Task (raw score); memory (immediate recall, delayed recall, and retroactive and proactive interference) 
will be measured using the Auditory Verbal Learning Test; delayed visuospatial memory and 
constructional praxis will be measured using the Rey-Osterrieth Complex Figure Test; attention will be 
assessed using the Paced Auditory Serial Addition Test (3- and 2-second tests) and forward and 
backward Digit Span measures from the WAIS-IV (raw scores). Executive function will be measured 
using the Trail-Making Test A and B and the Stroop Test. The Wide Range Achievement Test (WRAT) 
will be used to assess premorbid intelligence and may be used as a covariate if necessary. 
We will use an additional measure of episodic memory since: 1) memory measures were most 
sensitive to effects of PA in our previous work [19], and 2) episodic memory is most sensitive to early 
stages of AD [20]. We will administer a primed cued-fragment completion task that has been used to 
assess the independent contributions of controlled and automatic memory processes in healthy older 
adults and individuals with very mild AD [21]. This measure is key for examining potential gains in 
memory ability conferred by the intervention, because age-related memory deficits often show the 
largest effects on the controlled aspect of memory performance [e.g., 22]. Thus, this measure would 
determine whether predicted gains from PA will have a selective influence on this aspect.  
To allow comparisons across studies, we will include measures from the NIH toolbox and measures 
in the IGNITE study. From the toolbox, we will include Dimensional Change Card Sort, List Sort 
Working Memory, Flanker, and Picture Sequence. To facilitate comparisons with IGNITE, we will use 
measures of logical memory, spatial relations, spatial working memory, and matrix reasoning from the 
WAIS-IV, and a measure of paired associate learning from the Virginia Cognitive Aging Project. 
All measures have been used extensively, have well-established psychometrics, are not reliant on a 
particular reading level, and have age-appropriate normative data adjusted for education level. These 
characteristics ensure that the measures are rigorous and reproducible. Normative data [23], 
assessment target age groups (NIH Toolbox measures), our preliminary data [24], evidence of change 
across the lifespan [25], and evidence of sensitivity to APOE4 status [26-28] suggest that we can 
expect variability in performance and that there will be room for improvement on these cognitive 
measures within this age group. For cognitive domains with 3 or more measures, composite scores will 
be used in the first level of analyses.  For all tests, alternate forms will be used when available.   
3c.4.4 Submaximal Aerobic Fitness. Aerobic fitness will be estimated using a submaximal 
exercise test. Participants will complete a submaximal exercise test on a treadmill while using a 
metabolic cart to collect expired gases. Changes in aerobic fitness will provide an indicant of the 
physiological responsiveness to the PA intervention. 
3c.4.5 MRI. The collection of MRI data will allow for the assessment of cerebral structure and 

function. These measures are expected to be sensitive to the PA manipulation [13, 29-31] and to 
APOE4 status [20, 32-37].  MRI exams will be conducted at the Gateway MRI Center. Images will be 
acquired on a Tim Trio Siemens 3T MRI Scanner with a 12 channel receive-only head coil. Images will 
be acquired parallel to the IGNITE trial and sequences will be written by Jung (consultant). Only 
participants who meet criteria for MRI will be scanned. Exclusion criteria include:  may be pregnant, 
have previous head or neck surgery, have any metallic or magnetic implants that are contraindicated 
for MRI, have had surgery in the last 6 weeks, or weigh over 450 lbs. 
3c.4.5a Acquisition. A high resolution T1-weighted image will be collected using a 3D 
volumetric MPRAGE sequence with 0.9 mm isotropic resolution (TR=1900ms, TE=2.93ms, TI=900ms, 
flip angle=9 degrees, 176 slices) to assess brain volumes. Images will be immediately re-acquired if 
artifacts due to motion or other sources are detected. Whole-brain connectivity will be assessed using 
blood oxygenation level-dependent (BOLD) imaging during resting state. Participants are asked to view 
a cross in the middle of a screen with eyes open. Images will be collected parallel to the anterior 
commissure-posterior commissure (AC-PC) line using multi-slice gradient-echo planar imaging (EPI) 
(TR=2000 ms; TE=40ms; field of view=24cm (frequency) x 15cm (phase); matrix size=96 x 86, 40 
slices, 3mm thickness, no skip; voxel resolution=3 X 3 X 3 mm).  Diffusion tensor imaging (DTI) will be 
collected to assess white matter microstructure with a dual spin-echo echo-planar imaging (EPI) 
sequence (30 directions, b=1000 s/mm2 TE = 90ms effective; NEX = 1, FOV=25.6, matrix 
size=128x128x40, slice thickness=3mm no gap; TR=5300ms). 
3c.4.5b Image Processing. All images will be processed using freely available software 
(Freesurfer, SPM, FSL) in a customized workflow. T1-weighted anatomical images will be processed 
using the Freesurfer longitudinal pipeline and recommended manual editing for optimization [38]. fMRI 
data will be aligned to the T1 data, then warped to template space by applying the transforms 
computed on the T1 data. Distortion correction will be performed using TOPUP in FMRIB’s Software 
Library (FSL) [39, 40]. To control for physiological noise, all data will be preprocessed to remove white 
matter and cerebrospinal fluid signal using the CompCorr method implemented through the Conn 
Toolbox [41, 42]. Fluctuations in signal remaining after controlling for head motion will be identified 
using ART software [42] and time points with meaningful deviations in signal will be regressed out. DTI 
data will be processed using FSL software. Tools from FMRIB’s Diffusion Toolbox will be used to 
correct for eddy current distortion and calculate diffusion tensors from which fractional anisotropy (FA) 
will be computed. FA maps generated from the DTI sequence will be normalized to standard space 
using SPM12.   
3.c.4.5c Analysis. Hippocampal volumes will be estimated using Freesurfer, including quality 
control and manual editing as recommended. Regions of interest will be extracted from other image 
formats using WFU Pickatlas [43] for inclusion in SEM models.  
3c.4.6 Covariates. Because cognitive performance is expected to be associated with age [e.g., 23, 
44, 45] and education [e.g., 23, 45], these variables will be included as covariates in the models.  
Identifying additional individual difference variables that discriminate levels of responsiveness to the 
intervention may provide important insights for subsequent research. Data on variables that have been 
identified as important in past research [46-50] will be collected and explored as potential covariates. 
These include: sex, blood pressure, smoking, alcohol use, medications and supplements, sleep quality, 
body mass index, menopausal status, hormone therapy use, diabetes, and cardiovascular risk factors. 
When assessing FH, detailed information regarding blood-related relatives suspected to have AD or 
diagnosed with AD will be collected. This will include relationship to participant, age at diagnosis, and 
method of diagnosis. We will also collect data on parents who have not been diagnosed with AD (age 
and health status currently or at time of death).  Because a FH+ that is maternal and with a younger 
age of onset [51] results in higher risk, these variables will be included as covariates in statistical 
analyses. Participants also will complete the International Physical Activity Questionnaire [52] at pre, 
mid, and post-tests (monthly for UCC).  
3c.5 Data Management, Quality Control, Analytical Plan, and Power Analysis 
Procedures described here contribute directly to the rigor and reproducibility of the proposed study, 

to ensure the quality of data recording and analysis and appropriate determination of sample size. 
3c.5.1 Data Management. Data will include hardcopy surveys and data collection sheets and 
results of cognitive testing, MRI scans, genotype, and blood assays recorded electronically. We will use 
subject ID #’s and remove names as soon as possible to ensure that 
participant confidentiality is maintained. All electronic data will be de-
identified, and managed using REDCap software. This system allows us to 
control data access at the individual level and apply other rules and 
constraints that promote data quality. Pre-test data from the first subjects 
will be examined to identify data gathering problems to be addressed 
immediately. The dataset will be cleaned using standard methods to identify 
impossible and improbable data [53] including frequency distribution checks 
for outliers and problems in data gathering or entry. Validity checks will be 
performed as recommended [53]. MRI structural and white matter integrity 
data will be assessed using Freesurfer, and CONN will be used for RSC 
analyses.  Data will be exported to SPSS and a codebook for variables will be developed. Linear 
growth curve (LGC) analyses will be conducted using MPLUS [54]. 
3c.5.2 Quality Control.  Standard Operating Procedures (SOPs) for cognitive testing, the PAC 
intervention, submaximal exercise testing, and blood draws are in place from our Phase I clinical trial 
(PAAD) [1].  Modifications to the SOP to reflect the addition of cognitive measures and updates to 
procedures will be made prior to participant recruitment. MRI SOPs will be developed by Etnier (PI) in 
close consultation with Hugenschmidt and Jung (Co-Is) and based upon the existing MRI SOPs from 
IGNITE to maximize compatibility with the IGNITE study.  Post-doctoral fellows will be trained to 
implement the SOPs.  Throughout the study, Etnier (PI) will regularly observe testing sessions for 
quality control relative to SOPs, Karper will regularly observe PAC sessions, and Labban (Co-I) will 
conduct regular checks of the data to ensure quality control. 
3c.5.3 Analytical Plan. The conceptual model driving our research is inherently a model of change: 
changes in PA lead to changes in cognitive performance. The current state of the art in statistical 
models for studying change is LGC analysis [55-57] which models trajectories of observed change as 
reflecting an underlying (“latent”) developmental process. LGC analysis is an ideal statistical tool for 
testing our conceptual model because it allows us to 1) quantify both mean and individual variation of 
pretest levels of our outcome variables (e.g., cognitive performance, neurological function/structure, 
and biomarkers), as well as mean and variance of change in those outcomes over the intervention 
period; 2) test whether the PA intervention (PAI) and APOE4 status predict latent trajectories of change 
in these outcomes; 3) determine whether change in cognitive performance is mediated by putative 
mechanisms; and 4) test whether APOE4 status moderates the mediational models.  
Figure 3a illustrates our conditional LGC model to test Specific Aims 
1 and 2. In this model, the intercepts capture participants’ initial status; 
whereas, the slopes capture their change over time. The path arrow from 
PAI to the slope growth factor is quantified as a regression coefficient 
which we will use to explore the main effect of PA, APOE4 status, and 
sex (not shown) on change in outcomes over the intervention. We do not 
expect sex to affect the influence of PAI on relevant outcomes, but we will 
control for sex in our analyses; and, if sex is a significant predictor, we will 
conduct additional exploratory analyses to guide future research. We do 
expect that participants assigned to the PAI will exhibit greater 
improvements in cognitive performance than will controls, but that APOE4 status will exert minimal 
direct effects on these behavioral measures. This expectation arises from data gathered in our previous 
study, which suggests that APOE4+ who are not yet exhibiting behavioral signs of cognitive decline 
should not be expected to demonstrate steeper rates of improvement on cognitive measures compared 
to non-carriers [24]. However, we do expect both PAI assignment and APOE4 status to impact changes 
in neuroimaging and biomarker outcomes. Therefore, we will also test whether changes in 
neuroimaging (Specific Aim 3) and biomarkers (Specific Aim 4) are mediators of change in cognitive 

performance (Figure 3b). Because putative mechanisms will be measured at pre-test and post-test 
only, we will estimate residualized change in these variables using univariate ordinary least squares 
regression. We will use the resulting change scores to test whether changes in these putative 
mechanisms mediate changes in cognitive measures. PAI assignment and APOE4 status will serve as 
predictors of slopes for cognitive measures, but will also be regressed on mechanism change scores, 
which will in turn be regressed directly on the slope growth factor. This new parameterization of the 
model will allow us to formally test the indirect effect of PAI assignment on cognitive outcomes, via their 
direct effects on the putative mechanisms. Finally, APOE4 status will be tested as a moderator of the 
association between PAI and mechanism change scores. The final parameterization of the model will 
thus be a test of moderated mediation, and is a logical extension of the previous model: APOE4 status 
moderates the association between PAI and change in mechanistic outcomes, which are in turn directly 
predictive of change in cognitive outcomes. We expect participation in the PAI will produce 
improvements in cerebral structure, cerebral function, and biomarkers, and that these improvements 
will lead to improved cognitive performance.  
The portions of Specific Aims 3 & 4 that refer to putative mechanisms as outcomes will be tested 
using hierarchical multivariate regression techniques. Covariates and pre-test scores will be entered 
into the models at steps 1 & 2, and group assignment and APOE4 status will be entered at steps 3 & 4. 
Finally, the PAI x APOE4 status interaction will be tested at step 5. Entering predictor variables in this 
fashion will allow us to assess the relative importance (adjusted R2 and change in R2) of each to the 
prediction of post-test neuroimaging and biomarker status.  
All dependent variables are continuous, and expected to be normally distributed. Strategies to 
address violations of model assumptions may include sensitivity analysis, and computation 
of bootstrapped standard errors and confidence intervals. Missing data will be handled within growth 
models using maximum likelihood estimation when assumptions that data are missing at random or 
completely at random are met. If not, alternative strategies to handle missing data will be employed, 
such as the Diggle-Kenward selection model [58] or pattern-mixture model [59]. 
3c.5.4 Power Analysis. We estimated sample size requirements using preliminary data from our 
previous study [24]. Coefficients on which we based our analysis included expected values for slope 
factors, regression estimates of association between predictors (PAI, APOE4 status) and slope factors, 
and reasonable estimates of associations of the PAI-by-APOE4 status interaction with slope factors. 
We conducted Monte Carlo simulations with 5000 replications, requiring estimate and variance bias of 
<10%, and coverage >95% [60], to estimate power for our target sample size of N=240 with attrition 
rates of 10-30%. An initial sample of 240, provided the assumed attrition rates, would have an 
approximate power of 0.97 to detect a mean slope factor of 0.10, power of 0.86-0.96 to detect 
parameter estimates of 0.075-0.10 for slope regressed on PAI and APOE4 status, and power of 0.82-
0.92 to detect interaction effect estimates of 0.065-0.10 for PAI-by-APOE4 status regressed on slope. 
3c.6 Potential problems, alternative approaches, and limitations 
Although we have recruited, enrolled, and retained 54 participants over 1.5 years in our previous 
trial, we could have challenges enrolling 240 participants (120 PAI, 120 UCC) over 4 years.  If so, we 
will increase recruitment efforts through face-to-face meetings with local support groups and at 
community centers, places of business, places of worship, and other relevant locations. Additionally, we 
will recruit from 2 additional adjacent counties (Caswell, Chatham) which would result in 32,594 
additional potential participants in the targeted age range. Lastly, we will focus on retaining participants 
by maintaining regular contact with the UCC and fostering a sense of belongingness and commitment 
by those in the PAC.  The focus on FH+ is purposeful and reflects our interest in understanding the 
potential of PA for these individuals. However, we recognize that the results may not generalize to 
persons who are FH-. Additionally, the use of a UCC was carefully considered. However, we 
acknowledge that with a UCC we cannot differentiate effects of the PAC specific or not specific to 
exercise. That is, participants in the PAC will be different from those in the UCC in more ways than 
simply the exercise itself (e.g., they will receive more regular attention, they will have social 
interactions, they will have a sense of accomplishment).  As such, if our hypotheses are supported and 
suggest that PA benefits cognition by persons with a FH+, future studies will be needed to identify the 

specific agents of change in the PAC causally linked to these benefits. 
3c.7 Rigor and Reproducibility 
Recognizing the NIH’s commitment to rigor and reproducibility, our study meets the highest 
standards [61, 62]. We have estimated sample sizes conservatively and considered expected dropout 
based on our previous Phase I clinical trial (PAAD).  We have strategies in place to handle missing 
data.  We will use random assignment and will ensure that research staff collecting cognitive, 
neuroimaging, and biomarker data will remain blinded to group assignment throughout the study.  We 
have procedures in place to ensure the fidelity and consistency of implementation of the PA program 
across sites. We also have procedures to provide quality control at the level of data collection, data 
analysis, and data reduction.  We will assess PA in the UCC group to assess possible cross-
contamination. We will use validated, well-established measures of cognition, commercially available 
assays of biomarkers, and currently accepted methods to collect, reduce, and analyze neuroimaging 
data. We will consider sex as a biological covariate and pursue its effects from an exploratory 
perspective if it is a significant predictor. Lastly, when we disseminate our findings, we will heed the 
reporting standards guidelines relative to issues of rigor and reproducibility [63]. 
3c.8 Timetable (Figure 4) 
Because this study extends our previous work, most SOPs are already in place; thus, we require 4 
months to finalize SOPs, obtain approval from the Institutional Review Board, train experimenters, 
prepare a REDCap data collection system, and finalize data collection materials. Anticipating 15% 
dropout and with a target of 200 completing the program, we will enroll 240 participants. Initially, we 
intended to recruit in cohorts and we used this model prior to the COVID-19 pandemic. Using this 
model, for each cohort, we planned to use 6-8 months (longer period allowed for the later cohorts and 
to minimize overlap of testing sessions across cohorts) for recruitment and screening. We would then 
use two months to complete pretesting. After pretesting, participants would be randomly assigned to 
conditions for the 12-month intervention which would include mid-testing and post-testing at 6-month 
intervals. We planned to ultimately enroll 4 cohorts (n=60 each). After the COVID-19 pandemic, were 
were no longer able to implement the exercise program at YMCAs. As such, we changed our 
recruitment model to strive for 10 participants per month with randomization occurring after 
approximately 20 people were tested. This allowed for exercise groups to start on a more rolling basis, 
for the catchment area to be increased due to the virtual exercise, and still allows us to meet our 
recruitment goals. The modified time table is shown below.  
 
Data reduction and preliminary analyses will be ongoing, but will move towards completion in the 
final 5 months of Year 5. We anticipate disseminating findings at national and international conferences 
based upon data collected during the trial as shown in Figure 4. We will publish a paper describing the 
trial in Year 2, papers on the pre-test data will be prepared in Year 4, and we will prepare manuscripts 
from the entire data set and plan future studies in Year 5. 
 
 
 

4.1 Protection of Human Subjects 
4.1.1 Risks to Human Subjects 
4.1.1.a. Human Subject Involvement, Characteristics, and Design.  
Volunteers will be recruited from six counties (Guilford, Forsyth, Randolph, Rockingham, Davidson, 
Alamance) in the Piedmont region in North Carolina and from other locations within 2 hours drive of 
Greensboro. The combined population of the six counties is 1,388,480, of whom approximately 469,588 
are in the target age range of 40-65 years [77]. Recruiting from these counties, a representative sample 
in this age range would be predominantly white (67%) and black (23%). Approximately 9% of the 
population are Hispanic or Latino and approximately 52% are women. 
 
Participants will be recruited using a multi-pronged approach which will include newspaper 
advertisements, radio announcements, social media, electronic newsletters, and posting of flyers. Local 
area agencies (please see Letters of Support) and the Trial Match service through the Western Chapter 
of the Alzheimer’s Association will also assist with recruiting. Participants from all ethnic groups will be 
included in the study. The representativeness of the sample with respect to race will be monitored 
periodically and, if necessary, additional recruitment efforts will be targeted towards underrepresented 
racial groups.  
 
The goal of the selection criteria is to include middle-aged English-speaking adults (40-65 years) with a 
family history of AD (FH+) who are cognitively normal, who are not otherwise clinically impaired, who 
are healthy enough for exercise, and who are identified as sedentary according to ACSM Guidelines 
[ACSM, 83]. Sedentary is defined as participating in physical activity at a moderate intensity for 30 min 
or more fewer than 3x/week over the last 3 months and is assessed by self-report. Participants who will 
be excluded are those who:  
o meet the criteria for clinical cognitive impairment (MCI, AD, or other forms of dementia) 
assessed in two stages. First, during the telephone screening, we will use the modified 
Telephone Interview for Cognitive Status (TICS-m). The TICS-m has acceptable sensitivity and 
specificity in the detection of dementia [110] and amnestic MCI [111] and does not suffer the 
same ceiling constraints as do other measures of cognitive impairment [111, 112]. Participants 
who score <36 will be excluded [111]. Second, at baseline screening, participants will complete 
the Montreal Cognitive Assessment (MoCA).  We include people scoring equal or higher than 
25 and exclude people scoring equal or lower than 22 without additional consideration. For 
people scoring 23 or 24, the MoCA-MIS cutoff (< 11) is additionally considered to screen out 
individuals with potential mild cognitive impairment (MCI). For people meeting both MoCA-TS (≤ 
22) and MoCA-MIS (≤ 10) may be close to MCI rather than normal cognition based on Kaur, 
Edland, and Peavy (2018)’s data. These stratified and double-layered criteria will maximize true 
positives and minimize false positives for the detection of MCI as distinct from normal cognition.  
o are unable to perform PA because have known cardiovascular, metabolic, or renal disease and 
are symptomatic (assessed using the 2014 PAR-Q+) or because of orthopedic limitations as per 
the Guidelines of the ACSM [112]. 
o self-report any history of confounding neurologic (e.g., traumatic brain injury, prior stroke, 
myelopathy, myopathy, peripheral neuropathy, brain tumors), psychiatric (e.g., active major 
depression, any history of schizophrenia or bipolar disorder), or active severe or functionally 
disabling neurologic or medical diseases (e.g., Parkinson’s disease, active treatment for 
cancer), or any other conditions that might limit exercise or pose a danger to the patient – 
assessed using a Medical Health History (MHH). 
o report the current use of medications to treat symptoms of AD or that adversely affect cognition 
(MHH). 
o Meet the criteria for depression (score >16 and anhedonia or dysphoria nearly every day for the 
past 2 weeks, plus additional symptoms in 2 or more other DSM symptom groups reported as 
occurring nearly every day for the past 2 weeks or 5-7 days in the past week) using the short 
form of the Center for Epidemiological Studies Depression Scale Revised (CESD-R). Report 

suicidal ideation on the CESD-R.  
 
According to the American College of Sports Medicine, individuals categorized as participating in 
regular exercise must perform “planned, structured physical activity at least 30 min at moderate 
intensity on at least 3 d*wk-1 for at least the last 3 mo” [11, p. 33].  We will be recruiting participants 
who do not meet this definition and hence would be categorized as “not” participating in regular 
exercise. As per the ACSM Guidelines [11], individuals who do not participate in regular exercise must 
be further categorized as a) having no diagnosis of cardiovascular, metabolic, or renal disease and no 
signs or symptoms suggestive of these diseases (apparently healthy); b) having known cardiovascular, 
metabolic, or renal disease, but no signs or symptoms suggestive of these diseases (asymptomatic); or 
c) signs or symptoms suggestive of cardiovascular, metabolic, or renal disease (symptomatic).  This 
determination will be based upon the medical health history and the participants answers to questions 
from the 2014 PAR-Q+.  As per the ACSM Guidelines: 
• 
apparently healthy individuals will be asked to provide physician consent, but can participate in 
the exercise program and exercise testing without medical clearance and can progress to 
vigorous intensity exercise (following ACSM Guidelines) 
• 
asymptomatic individuals will be required to receive medical clearance from their personal 
physician prior to being allowed to participate in the exercise program or exercise testing.  
These individuals may perform light to moderate intensity exercise and may be progressed to 
vigorous intensity exercise as tolerated (following ACSM Guidelines) and as permitted by their 
physician. 
• 
symptomatic individuals who have managed their disease (stable) will be required to receive 
medical clearance from their personal physician prior to being allowed to participate in the 
exercise program or exercise testing. These individuals would likely require a diagnosis related 
to their specific symptoms. If these individuals were prescribed medication, they would have to 
be on the medication for 3 months and show signs of stabilization before being permitted to 
enroll in the study.  Once enrolled, these individuals may perform light to moderate intensity 
exercise and may be progressed to vigorous intensity exercise as tolerated (following ACSM 
Guidelines) and as permitted by their physician. Symptomatic individuals who do not have good 
disease management (unstable) will not be allowed to participate. 
 
To maximize adherence, we will take the following steps which have been identified as important: 1) 
use a group-based program whereby participants complete the program in a cohort; 2) offer incentives 
for adherence to the PA program; and 3) provide compensation for each testing session. 
 
4.1.1.b. Sources of Materials 
The research materials will be obtained from identifiable living human subjects in the form of blood 
samples, saliva samples, physical measurements, questionnaire data, neuroimages, and cognitive 
performance data.  
 
4.1.1.c. Potential Risks   
All of the testing procedures used have minimal associated risks.  
Cognitive testing and questionnaires:  There are no known potential risks involved with 
completion of the cognitive testing or completion of the questionnaires. However, some participants 
may be identified by the research staff as meeting the criteria for clinical cognitive impairment or for 
depressive symptoms. These participants will contacted by Dr. Tomika Williams (Gerontological 
Primary Care Nurse Practitioner) who will encourage them to schedule an appointment with their 
personal physician.  
Blood draws:  Risk of infection is minimal with the blood draws. Only slight discomfort should 
occur when the blood is drawn and the pain should be approximately equivalent to a mosquito bite. 
Bruising may occur following the blood draw and may result in mild-to-moderate soreness to the touch 
for several days.  
Exercise program and submaximal fitness tests:  As described in 4.1.1.a, all participants will be 

considered non-exercisers by our inclusion criteria and we will further identify participants as apparently 
healthy, asymptomatic, or symptomatic based on ACSM guidelines [11]. Those who are apparently 
healthy can safely participate in moderate-to-vigorous intensity exercise (as will be used in this 
intervention and in the exercise testing) according to the ACSM guidelines. Those who are 
asymptomatic, must provide evidence of medical clearance for participation, but then may participate in 
moderate intensity exercise and progress to vigorous intensity exercise as tolerated and as permitted 
by their physician. Those who are symptomatic, must provide evidence of medical clearance for 
participation and must show that their symptoms have stabilized, but then may participate in moderate 
intensity exercise and progress to vigorous intensity exercise as tolerated and as permitted by their 
physician.  Those who are symptomatic and unstable may not participate. For those who participate in 
the PA intervention, risks associated with participation are minimal and include:  (a) muscular fatigue 
and/or muscle soreness during and after exercise and (b) risk of falling while exercising. As with any 
type of exercise, there is a very minor risk of injury as abnormal changes in heart function may occur 
and, in extremely rare instances, heart attack may occur. However, the risk of this happening is 
reported as 1 in 10,000 deaths during maximal exercise tests and risks are dramatically lower for the 
submaximal physical activities and submaximal fitness tests that will be used in this program.   
In response to COVID-19, additional safety precautions have been implemented. Anyone who 
has been diagnosed with COVID-19 will be required to obtain physician consent to complete the 
submax test. Exercise intervention - (NOTE: For b and c below, if their physician advises them to wait 
longer than the 2 weeks, that will supersede our guidance) a). Participants who have been diagnosed 
with COVID-19 and hospitalized will be required to obtain physician consent to resume participation in 
the exercise program. Once approved, they will begin with very light intensity, will be encouraged to 
self-monitor physiological responses, and will only be progressed gradually. b). Participants who have 
been diagnosed with COVID-19 and who experience mild/moderate symptoms that do not require 
hospitalization will not resume exercise until 2 weeks after cessation of functionally-limiting symptoms 
(e.g., fever, shortness of breath, fatigue). At that point, they will begin with light intensity, will be 
encouraged to self-monitor physiological responses, and will only be progressed gradually. c). 
Participants who have been diagnosed with COVID-19 and who experience no symptoms or very mild 
symptoms will be allowed to resume exercise at their discretion at a reduced or light intensity, but will 
be encouraged to self-monitor physiological responses to ensure symptoms do not appear. 
 
Magnetic Resonance Imaging:  There are no known risks associated with having a magnetic 
resonance image as long as requisite safety precautions are taken. The precautions that must be taken 
are with respect to metal objects (external and internal), potential for burns, potential for hearing loss, 
muscle twitching and tingling, and risk for pregnant women. We will use a 2-step screening protocol, 
explicit instructions, and constant availability for communication during the scans as ways to further 
minimize these risks (see 4.1.2.b). Furthermore, participants will be informed that the MRI images 
completed at our facility are part of a research study and are not for clinical diagnostic purposes. They 
will be told that MRI images in this study will not be reviewed by a physician, but they will be given the 
option to receive a free copy of their images on a CD.  Participants will be told in advance that although 
the scan is not for diagnostic purposes, program staff will notify them if they see a substantial deviation 
from normal anatomy. If they indicate that they would like to be notified, Dr. Tonya Williams 
(Gerontological Primary Care Nurse Practitioner) will contact the participant, we will provide him/her 
with a free copy of their images on a CD, and we will suggest that they contact their physician for follow 
up.  If they indicate that they would not like to be notified, we will not alert them to the deviation.  
Participants will be regularly reminded that the research team cannot diagnose conditions. 
 Genotyping:  Similar to our previous study, participants will be informed that based upon ethical 
considerations and in compliance with recommendations [64], they will not be given information about 
their genotype. Additionally, research staff will explain to participants that their privacy will be protected 
and the confidentiality of their data will be maintained by insuring that their genetic sample will be 
identified by ID# only. Genotype information, linked only to an ID#, will only be made available to data 
analysts and linked only to an ID# and will not be made available to intervention or research staff who 
interact directly with participants. If participants were to become aware of a heightened genetic risk for 
Alzheimer’s disease, this might result in a negative emotional response. However, we will take the 

necessary steps to insure that participants do not gain access to their genotype information (see 
4.1.2.b). 
  
4.1.2. Adequacy of Protections against Risks 
4.1.2.a. Recruitment and Informed Consent  
Participants will be recruited through newspaper advertisements, radio announcements, social media, 
electronic newsletters, and posting of flyers. Local area agencies (please see Letters of Support) and 
the Trial Match service through the Western Chapter of the Alzheimer’s Association will also be used 
for recruiting. Interested participants will be asked to contact staff by telephone. Research staff will 
describe the study procedures. Those who remain interested will complete the TICS-m and answer 
questions to ascertain initial eligibility (e.g., who in your family has been diagnosed with Alzheimer’s 
disease, what is your age). Because participants will not yet have been consented, none of this 
information will be recorded in an identifiable fashion (i.e., only descriptive information will be recorded 
such as xx persons were excluded because of hearing difficulties). Eligible participants will be 
scheduled for pre-testing. Following the telephone interview, participants will be contacted by the 
Cognitive Testing post-doctoral fellow to complete additional surveys (e.g., the Medical Health History 
[MHH], the CHAMPS, demographics, CESD-10, PSQI). At the pre-test, informed consent will be 
obtained, additional screening will be performed using the Montreal Cognitive Assessment (MoCA), and 
a saliva sample will be obtained. Participants will be told about all aspects of the study including that 
their genotype information will not be shared with them and that blood samples, imaging data, and 
genetic material collected during the study will be stored by ID# and kept indefinitely to allow for future 
analyses. If a participant does not want his/her blood sample, imaging data, and/or genetic material to 
be stored for future analyses, he/she will be able to opt out of this aspect of the study while remaining 
eligible for all other aspects of the study. Participants will be asked to read the consent form and 
encouraged to ask questions before signing. A copy of the signed consent form will be offered to the 
participant, and the original signed copy will be kept in the participant’s secure file. We will take the 
following steps to maximize our chances of following subjects after 1-year:  1) include follow-up contact 
provisions in the consent form, 2) maintain contact with subjects about study progression   
4.1.2.b. Protections against Risk  
Exercise program and submaximal fitness tests:  To ensure participant safety, all physical activity 
sessions will be administered according to the guidelines established by the American College of 
Sports Medicine [ACSM, 83].  As described in 4.1.1.a, participants will be identified as apparently 
healthy, asymptomatic, or symptomatic and we will follow ACSM Guidelines with respect to these 
qualifiers. We have also implemented safety protocols relative to a person being diagnosed with 
COVID-19. Although the risks of an adverse event during or in response to exercise are low when 
following ACSM Guidelines, we will take additional safety precautions.   All staff involved in the PA 
program or in exercise testing will be trained in procedures to handle emergencies or urgent problems 
and trained in basic cardiopulmonary resuscitation (CPR).  As an added safety precaution, an 
automated external defibrillator (AED) will be available.  
 
Blood draws:  Although the risk is minimal, infection is possible when blood samples are taken. The risk 
of infection will be minimized through the use of sterile techniques by a trained technician. Additionally, 
because blood and saliva samples are biohazards, all OSHA related guidelines will be followed 
regarding training of research staff and the disposal of biohazards. 
 
Exclusion criteria:  Participants will not be informed of their cognitive performance; however, if a 
subject’s cognitive performance is judged to be of concern (e.g., they do not meet the criteria for 
participation), the participant will be contacted by Dr. Tomika Williams (Gerontological Primary Care 
Nurse Practitioner) who will encourage the participant to consult with his/her personal physician. 
Similarly, if a participant’s score on the Center for Epidemiological Studies Depression Scale – Revised 
(CESD-R) suggests that he or she is experiencing depressive symptoms that might be indicative of 
clinical depression or suicidal ideation, the participant will be contacted by Dr. Tomika Williams 
(Gerontological Primary Care Nurse Practitioner) who will encourage the participant to consult a 
physician.  

 
Magnetic Resonance Imaging (MRI): 
MRI is a non-invasive technique that does not have any known risks once necessary safety 
precautions have been taken. The precautions must be taken with respect to metal objects, potential for 
burns, potential for hearing loss, muscle twitching and tingling, and risk for pregnant women.  The 
following steps will be taken with regard to each of this risks. 
 
Metal objects: Metal objects within the body or on/in clothing can cause harm to participants, in 
addition to distorting the quality of the MRI images. Participants will complete the MRI Recruitment 
Screening Form and the Gateway MRI Screening Form and will go through an extensive screening 
process to determine if it is safe for them to have an MRI exam.  In addition, such things as keys, 
watches, and credit cards will be kept safely away from the machine. We will ask participants to take off 
all removable metal (e.g. jewelry, piercings, etc.). People with devices or objects inside the body that 
are affected by strong magnetic fields (i.e. metallic foreign bodies inside a person's head or eyes, 
incompatible medical implants, pacemakers, brain stimulators, blood vessel clips, etc.) will not be 
allowed to participate under any circumstances. Knowingly participating in this study with these types of 
metallic implants can lead to serious injury or death. Although metal objects sensitive to strong 
magnetic fields are not allowed in the MRI scanner, there are many metal objects that are not sensitive 
to strong magnetic fields, such as dental work, pins or screws used during surgery, and even some 
tattoos contain metal. People with these types of metal objects may safely participate in this study.  
   
Burn risks: In extremely rare cases, metal in the body (e.g., in tattoos) exposed to the powerful 
radio waves used in MRI may heat up. This heating occurs gradually but if it goes unreported during the 
MRI exam it could lead to burns. Such burns are easily prevented by reporting any heating sensations 
to the technologists immediately. For safety, participants will be monitored the entire time you are in the 
scanner. The study team will be able to communicate with participants during the exam through an 
intercom. Participants will also be given a ball to squeeze to signal the researcher to stop the exam 
immediately and for any reason. 
 
Hearing loss: MRI scanners when taking a picture are very loud. Participants will be required to 
wear Comply Canal tip earplugs during the exam. When the earplugs are used properly, the noise from 
the MRI scanner is as loud as a garbage disposal or food blender. If the earplugs are not inserted into 
the ear canal then temporary hearing loss is possible. If at any time the noise from the MRI machine is 
too loud, participants will be instructed to inform the technologist. 
 
Muscle twitching and tingling: MRI machines turn magnetic fields on and off very quickly to make an 
image. In rare cases, this may cause a person's muscles to twitch and tingle. The muscle twitching and 
tingling are temporary and will stop as soon as the scanner stops. In some rare cases, some individuals 
find the muscle twitching and tingling to be uncomfortable and cannot continue with the MRI exam. If 
this happens, participants will be instructed to let us know and will be released from the study. 
 
Pregnancy: It is unclear at this time whether strong magnets are a risk to unborn fetuses. Due to 
the unknown risk and potential harm to an unborn fetus from any MRI scan, pregnant women will be 
excluded. All women in this study are between the ages of 40-65 and are unlikely to be pregnant, but 
we will ask this question before entering the scanner. 
 
Data confidentiality and genotyping 
All subjects’ data will be coded so that the identities of the participants are not contained on the 
processed data. All data collected in this study will be identified only by subject number, and not by 
name, and data will only be reported in anonymous or aggregate form. Further, saliva samples are 
coded based on de-identified information (i.e., subject ID #) so that the WFU lab processes DNA solely 
on the basis of the ID # and has no access to subject information which is kept by the research staff. 
Every effort will be made to keep all information confidential. If data are used for publication, no 
identifying information will be included. The subject name-code number logs will be kept in a locked 

cabinet in the Physical Activity and Cognition laboratory at the University of North Carolina Greensboro. 
All procedures and data handling will be in compliance with HIPAA regulations. 
 
In addition, researchers will request a Certificate of Confidentiality (CoC) from the National Institutes of 
Health.  Information about this CoC will be included in the informed consent. The CoC will allow the 
researchers to protect participants’ privacy. The researchers can use the Certificate to legally refuse to 
disclose information that may identify participants in any federal, state, or local civil, criminal, 
administrative, legislative, or other proceedings, for example, if there is a court subpoena. The 
researchers will use the Certificate to resist any demands for information that would identify the 
participant [except as explained below]. 
 
The Certificate cannot be used to resist a demand for information from personnel of the United States 
federal or state government agency sponsoring the project and that will be used for auditing or program 
evaluation of agency funded projects or for information that must be disclosed in order to meet the 
requirements of the federal Food and Drug Administration (FDA). Participants should understand that a 
Certificate of Confidentiality does not prevent the participant or a member of your family from voluntarily 
releasing information. If an insurer, medical care provider, or other person obtains the participant’s 
written consent to receive research information, then the researchers will not use the Certificate to 
withhold that information. 
 
The Certificate of Confidentiality will not be used to prevent disclosure to state or local authorities of 
reports of harm to self or others. 
 
To minimize burden, all data collection sessions will be held in a flexible manner to accommodate 
potential limitations in endurance and abilities. Thus, testing will be discontinued or postponed as 
needed. To protect against attrition due to participant burden, the investigators will monitor ongoing 
participation in the study, contact individuals who appear at risk for dropping out, and provide support 
and encouragement. At all times, they will be encouraged to call with questions or concerns regarding 
their participation in the study. Participants will be encouraged to seek out medical advice relative to 
any problems that are detected. Of course, participants may withdraw from the study at any time. 
  
4.1.3. Potential Benefits of the Proposed Research to Human Subjects and Others 
The small risks incurred by the participants are outweighed by the potential benefits of this research. 
Participants randomly assigned to the physical activity condition may benefit directly as a result of 
participation in the physical activity program which we expect to produce cognitive benefits and other 
psychological and health benefits. Participants randomly assigned to the usual care control condition 
will be given a short-term YMCA membership upon completion of post-testing.  The general information 
that will come from this study may provide evidence that the physical activity program can improve 
cognitive performance in middle-aged adults with a family history of AD.  Since the risks associated 
with participation are minimal, the potential cognitive, psychological, and health benefits to participants 
outweighs the risks.  
 
4.1.4. Importance of the Knowledge to be Gained  
The findings of this study are important because they will advance our knowledge regarding the efficacy 
of this physical activity intervention for middle-aged adults with a family history of Alzheimer’s disease. 
The findings of this study will contribute to our understanding of the use of this physical activity program 
as a preventative behavioral intervention that protects the cognitive abilities of middle-aged adults.  
 
4.1.5 Data and Safety Monitoring Plan 
We have developed an internal data and safety monitoring plan (DSMP). The DSMP will be 
implemented through a). communication with and oversight by Safety Officers (Dr. Maryjo Cleveland, 
Director of Geriatric Consultation Clinic, Wake Forest School of Medicine; Dr. Jeff Williamson, Program 
Director, J. Paul Sticht Center for Healthy Aging and Alzheimer's Prevention) and the Primary 

Investigator (PI, Dr. Jennifer L. Etnier); b). training of research staff; c). monthly conversations with the 
Safety Officer, the PI, Dr. Tomika Williams (Adult Gerontological Primary Care Nurse Practitioner), 
relevant co-investigators, and other staff; d). confirmation that no one working on the research funded 
by this grant has any financial conflict; e). communication with and oversight by the Office of Research 
Compliance (ORC) at the University of North Carolina at Greensboro (UNCG); and f). communication 
with the NIA Program Administrator. The primary purpose of the plan is to allow us to monitor safety 
during the study with secondary charges related to the data collection procedures and the collection of 
efficacy data.  These aspects of the implementation of the DSMP are described in further detail in the 
Data Safety Monitoring Plan. 
 
Although we are aware of the potential for serious adverse events (SAE) to occur with any type of 
moderate exercise training, it has been clearly documented that the subjects of the type we will be 
working with can safely engage in exercise training   Even though the risk of a SAE is very low, all 
personnel who supervise the exercise program or who conduct submaximal fitness tests will be trained 
in Cardiopulmonary Resuscitation (CPR). Further, all personnel working in the neuroimaging facility will 
receive appropriate safety training and certification. With an eye to insuring the safe conduct of the 
study we will review all safety and emergency procedures on a routine basis.  As a matter of policy, all 
study personnel will participate in Responsible Conduct of Research (RCR) education programs. 
 
 
  
 
 
 
 
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Etnier, J.L., et al., Cognitive performance in older women relative to ApoE-ε4 genotype and aerobic fitness. Med Sci 
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Caselli, R.J., Recruiting efficacy for ApoE genotypes, J.L. Etnier, Editor. 2008: Greensboro, NC. 
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