Influence of Exercise on Neurocognitive Function in Breast Cancer
Phase: NA
Conditions: Breast Cancer
Sponsor: University of Pittsburgh

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Protocol & Statistical Analysis Plan 
Official Title: Influence of Exercise on Neurocognitive Function in Breast Cancer 
NCT02793921 
Document Date: 6/14/2023

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Title of study: 
Exercise Program in Cancer and Cognition (EPICC) 
 
 
Brief description: 
This is a single-site randomized control trial to test whether a moderate-intensity aerobic exercise 
intervention can prevent cognitive decline in postmenopausal women with breast cancer. Women who 
meet the eligibility criteria will be randomly assigned to either receive the intervention or usual care. Data 
will be collected prior to initiating endocrine therapy for breast cancer (TP1) and approximately 7 months 
later (TP2)and include fitness testing and a battery of neuropsychic tests, blood biomarkers, and other 
behavioral measures; a subset of women will undergo MR imaging. For women who agree to additional 
follow-up, data will also be collected approximately12 months post TP1 (TP3).  
 
Specific aims: 
We will examine whether a well-controlled and monitored site-based exercise intervention improves 
cognitive function in postmenopausal women with early stage breast cancer.1. Compared to usual care, 
examine whether the 6-month exercise intervention improves cognitive function in postmenopausal 
women with early stage breast cancer. Hypothesis 1. Exercise will improve cognitive function in women 
receiving AI therapy in a domain specific fashion such that attention, executive and memory functions will 
be influenced more than other domains. 
 
2. Compared to usual care, examine the direct effects of exercise on neuroimaging metrics of brain health 
including regional gray matter volume, white matter architecture and functional dynamics of the brain and 
the pro-inflammatory biomarkers (IL-6, TNF-α), and explore the direct effects of exercise on symptoms 
(fatigue, sleep problems, depression, anxiety). Hypothesis 2. Exercise will improve neuroimaging metrics 
of brain health and pro-inflammatory biomarkers. 
 
3. Compared to usual care, explore whether the effects of exercise on cognitive function are mediated by 
a) neuroimaging metrics of cognitive function, b) IL-6 and TNF-α levels, c) symptoms (fatigue, sleep 
problems, depression, anxiety), d) and moderated by E2 levels over the first six months of AI therapy. 
 
Inclusion criteria: 
1. Postmenopausal women, defined by lack of a menstrual period for an entire year or confirmed by 
physician 2. 18-80 years of age 3. Able to speak and read English 4. Completed a minimum of 8 years of 
education 5. Diagnosed with stage 0, I, II, or IIIa breast cancer, confirmed by medical record 6. Within 2 
years post-completion of primary treatment 7. Will be at least two weeks post-breast conserving surgery 
or three weeks post-breast conserving surgery with sentinel lymph node biopsy or four weeks post-
mastectomy at the start of participation.  
 
 
Exclusion criteria: 
1. Prior treatment with cancer chemotherapy, central nervous system radiation, of intrathecal therapy 2. 
Prior cancer involving the central nervous system 3. Clinical evidence of metastatic disease 4. Any of the 
following breast cancer surgery complications unless approved by the participant’s health care provider: 
persistent seroma requiring aspiration, wound dehiscence, infection, prolonged drain output, 
lymphedema5. Reconstructive surgery unless approved by the participant's health care provider6. Any 
significant medical condition that would preclude them from exercising (e.g., uncontrolled diabetes, 
congestive heart failure, angina, uncontrolled arrhythmia or other symptoms that indicate increased risk 
for an acute cardiovascular or 
 
 
Research activities: 
Participants: We plan to recruit up to 350 postmenopausal women with early-stage breast cancer: up to 
127 will undergo an exercise intervention in this 6-month study described below and up to 127 will be 
enrolled in an usual care arm. A subsample of women who are randomized will participate in the 
neuroimaging component of the study(n=150; 75/group). 

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Locations: Recruitment will take place at St. Clair Budway Surgical Associates and Magee-Women’s 
Hospital of UPMC, Comprehensive Breast Care Center at UPMC Passavant-Cranberry, UPMC South 
Hills, UPMC McKeesport and Magee Woman care Center in Monroeville. Data collection will take place in 
participants’ homes, at the University of Pittsburgh School of Nursing and Sennott Square or Healthy 
Lifestyle Institute offices in Oak Hill. The exercise intervention will take place at Sennott Square, Baierl 
Family YMCA, Sampson Family YMCA, and JCC South Hills Branch or Healthy Lifestyle Institute offices 
in Oak Hill; alternatively, a participant may exercise in her home or other location of her choosing. MRI 
Imaging will take place at Carnegie Mellon University SIBR.  
 
Informed Consent: Written informed consent will be obtained from the participant prior to any study-
specific activities either in person or remotely.  
 
Assessments: Participants will complete a series of assessments at two timepoints; Time 1(baseline) will 
occur up to 2 years post-completion of primary treatment and before randomization and initiation of the 
exercise intervention/usual care. Time 2 will occur approximately within 1 month of completion of the 
intervention (or 7 months post-Time 1). Data collection at each timepoint will be composed of behavioral 
assessments, blood samples, neuroimaging and objective measures of physical activity and fitness. We 
will also objectively evaluate physical activity at 3 time points during participation (approximately every 5 
weeks).For participants who agree to additional follow-up via the addendum consent form, Time 3 will 
take place approximately 6 months post Time 2 and will consist of blood work, fitness testing and 
behavioral testing as described below. We will also objectively evaluate physical activity at midway (3 
months) between Time 2 and Time 3 as well as at Time 3. Participants will also be asked to maintain their 
edema cap and REDCap diaries during this time. Participants may be asked to complete assessments in 
two separate sessions at both TP1 and TP2.During one visit (which includes fitness testing), blood 
samples (approximately 30ccor 6 teaspoons) will be collected for biobanking for future evaluation of the 
relationships of biomarkers (including genetic biomarkers) with cognitive function, exercise, brain health 
and symptoms. Blood samples will be transported immediately, under conditions appropriate for 
preservation of the blood, and stored before the end of the day of testing. Samples will be maintained in a 
-80°C freezer in the Victoria building under the control of the principal investigators of this research 
project and will be maintained indefinitely. In the event that we need to send samples to a different facility 
outside of the University of Pittsburgh for analysis in the future, no identifying information will accompany 
the sample. Participants will be asked questions prior to the blood draw to determine any factors which 
may affect the findings. If a recent illness is noted, or certain other factors as noted on the form, are 
found, the participant may not be eligible for a blood draw at this visit. In this event, we will attempt to 
collect the blood at another time, either during a subsequent visit or other agreed upon time. Fitness will 
be objectively measured at Time 1 and Time 2. This test takes 15-30minutes to complete. Subjects will be 
given exercise testing instructions regarding proper clothing and footwear and will be told to abstain from 
caffeine or nicotine 2hours prior to the physical activity/fitness assessment. When participants report to 
the laboratory (Sennott Square) for the fitness assessment they will first be acquainted with the 
equipment, allowed to ask any questions, and get comfortable with walking on a treadmill. Before the test 
begins, the participant will get familiar with wearing the mouthpiece and nose clips or facemask required 
during the test in order to measure cardiovascular fitness. This helps to reduce anxiety associated with 
exercise and fitness testing. Cardiovascular fitness testing consists of progressively increasing exercise 
intensity while the subjects exhaled air is collected via a mouthpiece that is connected to a gas exchange 
chamber via a breathing tube. A comfort fitted head gear supports the mouthpiece. This reduces the effort 
of the participant to keep the mouthpiece in the correct position throughout the test. Alternatively, the 
participant may wear a facemask. The gas chamber that is being used is the Parvo Medics True one 
2400 metabolic cart, which is one of the most widely accepted systems in scientific research. Next we will 
provide the participant with adequate warm-up and stretching which will be monitored by the exercise 
physiologist. After 5-10 minutes of warm-up, the participant will walk on a motor-driven treadmill at a 
constant speed with increasing increments of the incline. We will conduct a submaximal vo2 fitness test 
which estimates the maximal oxygen capacity for the individual without reaching an exhaustive level. The 
sub-maximal graded exercise test is a modified Balke protocol in which the speed remains constant and 
grade is increased 1% each minute. This protocol has been used extensively in the literature for fitness 
training for both normal, at-risk, and impaired populations. Verifying maximal vo2 is difficult in at-risk 

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populations and therefore submaximal vo2 is often the desired method. This method has people walk at a 
slow - moderate pace with increasing grade increments up to the goal heart rate of 85% of the age-based 
maximum (220-age) or a rating of perceived exertion equal to or greater than 15 for those individuals 
whose heart rate response is blunted due to medication. This approach does not require the attendance 
of a physician because it is within the safe heart rate work zones andic leads do not have to be attached 
to the participant. The submaximal vo2assessment usually takes about 15 - 30 minutes to complete 
depending on the starting fitness level of the participant. During the exercise test, heart rate in 
continuously monitored along with blood pressure readings every two minutes. The blood pressure is 
obtained by the Sentech Tango 2 automated unit. This blood pressure unit is designed for exercise 
testing and has been tested as a valid and reliable system to measure blood pressure during exercise. 
When the subject reaches the endpoint goal of the exercise test, the mouthpiece and head gear 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 undergo 
a passive cool-down session in which they are seated in a chair. The participant's heart rate and blood 
pressure will be continuously monitored through the cool-down period and the participant will not be 
allowed to leave until the vital signs return to normal patterns. Blood pressure will also be monitored both 
before and after the fitness test to ensure that changes in blood pressure resulting from exercise are all 
normal. If the participants vital signs remain elevated after a cool down and rest period, the study 
physician will be called and will provide instructions for further monitoring of the participant. Situations like 
this are very rare and occur in less than 1/10,000 instances of fitness testing. These assessments will be 
conducted in Dr. Erickson’s laboratory by an exercise physiologist with >15 years of coaching and 
exercise testing who is AED and Recertified and has certifications in exercise physiology from the 
American College of Sports Medicine. Prior to engaging in exercise, participants may be fitted with one or 
two physical activity monitoring devices (Body Media Sense Wear mini armband or Autographing). The 
Body Media Sense wear Armband is worn around the upper left arm. Hectograph Link is a wristband 
worn on the non-dominant arm. Both devices will record physiological data (e.g., heat flux, sweat rate, 
etc.) over the duration of their exercise test; this information is used in an algorithm to determine intensity 
and amount of physical activity. The participants will be provided with detailed instructions regarding 
wearing of the device(s) as well as the option to remove the device(s) if it becomes problematic. 
Participants may also be asked to wear the physical activity monitoring device(s) for a week before or 
after the exercise testing session. This way we can get a more comprehensive idea of both activity and 
fitness levels. In addition, all participants, regardless of group assignment, will be asked to wear the 
device(s) 3 additional times through their participation in this study(approximately every 5 weeks) and 
again at the follow-up assessment (TP2) for total of 5 times over the course of participation in the study. 
Participants who wear the Actigraphy Link will be given a log at the time they receive their device. This 
log is used to report any time/reason the device is removed as well as information about their sleep. The 
self-report information contained in the log will be compared to and used in interpreting the device data. 
Alternatively, in the event that a participant cannot come to Sennott Square for this testing for any reason 
(e.g., preference, weather, pandemic-related restrictions.), a brief heart-rate monitoring session may take 
place virtually either by phone or teleconferencing. During this guided session, the participant will be 
asked to wear study-provided heart rate monitor for approximately 5 minutes and asked to report 
readings to study staff. Participants will also be asked to report their weight; home scale may provided for 
participants to use (with instructions to return via pre-paid mailer when complete). Data from the session 
can provide information to estimate results from a VO2 test. Participants may also be asked to complete 
questionnaires regarding exercise knowledge and self-efficacy during the physical activity and fitness 
assessment, time permitting. Alternatively, these may be included in the behavioral assessment. The 
behavioral assessment includes a battery of standard clinical neuropsychological tests that assess verbal 
ability, visuospatial ability, attention/processing speed, memory, and executive function, will be 
administered. These tests have standard administration protocols and will be administered by trained 
staff. This is expected to take approximately 90 minutes. We will also administer measures of 
demographics, mood and quality of life. The tasks and measures will include those listed in section 2.8. 
All of the tests will be identified by the subject ID and not by name. At times these tests can be mentally 
fatiguing or frustrating. Subjects will be told that the tests are designed to be challenging and that they 
should just perform to the best of their abilities. They will also be offered breaks as needed between tests 
to help reduce fatigue and frustration. Data collected will be transported by the research personnel 
immediately following the assessment if conducted in the participant's home to the office of the PI in the 

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Department of Nursing for locked storage. Alternatively, neuropsychological tests may be administered 
virtually via teleconferencing or phone as needed. Other measures may be administered via Redcap or 
by phone or teleconferencing as needed in the event that the participant cannot be seen in-person. 
Participants' location data (e.g., zip code or census tract) may be used to gather information from publicly 
availably datasets to provide information about community/neighborhood characteristics/social 
determinants of health (such as deprivation, safety, degree of segregation). 
 
Physical Activity Diary: All participants will be asked to provide information related to their physical 
activity throughout the study. Participants will be offered the option of a paper diary(uploaded in section 
2.8) or to complete a weekly electronic mail survey administered via REDCAP.  
 
eDEM Cap: All participants will be provided with the electronic Drug Exposure Monitor (Edema) to 
measure adherence to hormonal therapy. The Eden consists of a bottle cap fitted with a microprocessor 
that records the date and time the cap is removed from a medication vial. It also records the duration of 
time that the cap is off the vial. Eachmonitor has sufficient memory to record and store the dates and 
times of 2000doses. The Eden will be collected at the time of the follow-up assessment. Neuroimaging** 
No new MRI participants will be consented after February 2020 ** A subgroup(N=150) will have the 
following neuroimaging protocol at times 1 and 2. Each session will last approximately 1 hour. Seventy-
five women from each group will be randomly sampled to participate in neuroimaging based on age and 
baseline executive function. On the day of a participant's scan, she will be asked to arrive 30minutes 
before the start of her scan in order to provide time to review and sign a separate CMU consent form, and 
to undergo final safety screening by a SIBR MRI technologist. For the sake of brevity, MRI sequence 
parameters have been omitted but can be found in our prior publications.68,75,78,132,133 Brain 
Morphology: High-resolution anatomical MPRAGE (1mm3 voxels, 256 slices) images will be used for 
morphologic and volumetric analyses. The Free Surfer pipeline using its longitudinal option will be 
employed to compute cortical and subcortical volumes, cortical surface area, and thickness. Intracranial 
volume (ICV) will be used as a covariate in analysis of brain morphology. Regional values that can be 
exported into statistical packages (e.g., SAS) will be calculated. Errors and omissions will be corrected 
according to standard guidelines. Hippocampal subfields: We will use a focal (.4 x .5x 2mm) T2-weighted 
sequence for hippocampal subfield segmentation146. Since the dentate gyrus and CA1 subfields are 
more frequently affected byexercise147,148 and the head of the hippocampus may be affected more than 
thetail,68 this will allow us to segment hippocampal subfields to determine regional specificity of the 
intervention. White matter imaging will be performed using diffusion-weighted sequence (50 directions; 
2.5mm isotropic voxels). Images will be corrected for head motion and eddy current artifacts. For each 
voxel the first three principle eigenvectors of water diffusion will be calculated using FSL, which will be 
used to calculate fractional anisotropy (FA), axial and radial diffusivity components. These maps will be 
transformed into standard space using non-linear normalization. We will also conduct fiber tractography, 
which identifies tracts between regions and will use computational methods to dissociate crossing white 
matter fibers withinvoxels.149 Finally, white matter lesions (see next section) will be removed from the 
calculation of white matter integrity. White matter hyperintensities (WMH): We will use a T2 FLAIR 
sequence and an automated segmentation method, which uses an algorithm for automated selection of 
WMH lesions. The segmented WMH are then localized to white matter tracts using the Johns Hopkins 
White Matter Atlas. Resting state analyses: We will employ standard BOLD sequences used by PI: 
Erickson and graph theory approaches. Prior to generating brain networks (see150), all images will be 
motion corrected and registered. To control noise,151 data will be preprocessed to remove white matter, 
and cerebrospinal fluid signal. Motion scrubbing will be performed.152 Heart rate will be used to correct 
for physiological confounds. The time series from each node will be compared to every other node using 
regression analysis, generating a correlation matrix. A threshold is applied to the matrix to retain only the 
strong connections and is maintained to represent connection strength.153 Once an adjacency matrix is 
generated, network attributes can be calculated for each node. The data will allow for the identification of 
changes in network topology that are associated with the intervention. Cerebral Blood Flow(CBF) will be 
measured with multi-phase pseudo Continuous Arterial Spin Labeling(pals) with background suppression 
and will be analyzed using BASIL. Task-evoked activation: We selected the n-back working memory 
paradigm because our preliminary data demonstrates that walking interventions improve performance on 
this task.154 The task also has high reliability and involves a broad network of brain regions including the 
PFC, ACC, hippocampus, and basal ganglia.155 Participants determine whether a lowercase letter 

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matches the letter on the previous trial (1-back) or the letter two trials previously (2-back). We will employ 
FSL software with standard preprocessing steps including motion correction, temporal filtering, and 
spatial smoothing and a sample-specific template for registration.75,156,157 We will also use 
Psychophysiological Interaction (PPI) analyses to examine changes in functional connectivity between 
regions during task performance. 
 
 
Randomization: After completing the baseline assessments, subjects may be randomized into one of 
two groups: (1) Aerobic Exercise Intervention or (2) Usual Care. Subjects will be randomly assigned via a 
computer using a minimization algorithm with equal allocation to one of two treatment groups: 1) exercise 
intervention, or 2) usual care. Use of a minimization algorithm will insure treatment balance on two three 
factors:1) age at study entry (<60, >60 to <70, >70 years) (since age is associated with reduced fitness, 
cognition, and brain measures), and 2) baseline levels of cognitive function, particularly executive function 
(based on the composite mean Z-score, <-0.5, > -0.5 to < +0.5, > +0.5), and 3) the time since primary 
cancer treatment (<= 6months, >6 months to 12 months). Minimization is a form of adaptive treatment 
allocation in which the probability of assignment to the treatment groups does not remain constant, but is 
determined by the current balance and/or composition of groups. In this study, the treatment assignment 
probability will be adjusted in response to the marginal distributions of categories of age at entry and 
baseline executive function such that the marginal distributions of these categories will be approximately 
equal between treatment groups. Treatment group assignment will be generated and stored in a secured 
database. Aerobic  
 
Exercise Intervention Group: An exercise physiologist with a Master’s degree in Exercise Science with 
experience in the proper execution and delivery of the exercise intervention in normal and at risk 
population while maintaining a safe environment will be the staff member responsible for the training of 
other staff to deliver the exercise intervention at each site. This individual will also be responsible for 
developing the exercise protocol and will manage the safety and consistency of the intervention across 
sites. Exercise staff will have completed a degree in Exercise Science or related field (e.g., kinesiology) 
and / or have a fitness certification from a reputable organization. All staff will have a minimum of 2 years 
experience in exercise supervision and prescription. The exercise intervention will be aerobic in nature 
and performed at a moderate intensity as monitored by heart rate monitors and ratings of perceived 
exertion(RPE); both will be monitored on-site by a trained member of the research team. Trained study 
staff will be present at each exercise session at all intervention sites. Occasionally, participants may have 
heart rate abnormalities which can make it difficult to obtain accurate readings via a heart rate monitor. In 
these situations, exercise staff will employ additional strategies to monitor exercise intensity as described 
below:1. Rating of perceived exertion (RPE) – this scale is used for all study participants. Itis a scale that 
runs 6 (no intensity) to 20 (maximal intensity). It is asked of participants and tracked every 15 minutes 
during exercise.2. Rating scale of breathlessness – this scale will only be used for the rare case of 
compromised heart rate response. This scale runs 0.5 (very, very slight breathlessness) to 10 (very, very 
severe out of breath). If a participant rates 4 and above, the intensity will be decreased.3. Talk Test – this 
will be used by exercise trainers at every session. For those with heart rate issues, the exercise trainer 
will proceed through the entire session with ahigh level of attention focused on this procedure. While the 
trainer is in conversation with the participant, the trainer will be attentive to the level of gasping for breath 
while talking by the participant. If the participant is struggling to hold or continue a conversation due to the 
level of work, the intensity will be decreased. Walking on a motorized treadmill will be the encouraged 
mode of exercise; however participants may use other equipment such as upright bikes, recumbent bikes, 
ellipticals and indoor walking track, etc. Participants will begin by exercising 10-15 minutes for 3 
days/week during the first two weeks of the program and gradually increase the duration for the following 
4weeks until they reach 40–50 minutes per session/3 days per week. This level is then maintained for the 
remainder of the 6 months. This falls within the American College of Sports Medicine (ACSM) guidelines 
for cancer patients and survivors.106These guidelines (2010) recommend a weekly volume of 150 
minutes of moderate intensity or 75 minutes of vigorous intensity exercise, or a combination of thetwo.117 
The rate of increase will be tailored based on each subject’s baseline cardiorespiratory fitness level and 
response to exercise. All exercise sessions start and end with a vital sign check and 5-10 minutes of 
stretching for the purpose of warming up and cooling down. Staff trained in exercise physiology and 
coaching will supervise all exercise sessions and closely monitor adherence, intensity, and safety. In-

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person exercise, as described above, will be the preferred method for intervention delivery. However, 
participants may also exercise at their home, or other location of their choosing, as needed. When 
exercising at home, participants will be in contact with a study trainer at least weekly. Participants will 
receive guidance and coaching related to their exercise sessions - either in real-time or other time as to 
be accommodating as possible to the research participants. Heart rate during exercise sessions will be 
monitored via study-provided monitors and will be reported to trainers by the participant. Participants may 
be provided with online resources/videos for exercise (such as Be Fit Pitt) or may be encouraged to use 
an exercise resources available to them (such as owned exercise equipment, gym memberships, indoor 
or outdoor walking, etc.) 
 
Usual Care Group: will not withhold physical activity or limit physical activity in the usual care group but 
instead will let them engage in activity in the same manner as if they were not part of an active 
intervention.  
 
 
Statistical Plan 
 
Randomization: Subjects will be randomly assigned via a computer using a minimization algorithm with 
equal allocation to one of two treatment groups: 1) exercise intervention, or 2)usual care. Use of a 
minimization algorithm will insure treatment balance on two factors: 1) age at study entry (<60, >60 to 
<70, >70 years) (since age is associated with reduced fitness, cognition, and brain measures), and 2) 
baseline levels of cognitive function, particularly executive function (based on the composite mean Z-
score, < -0.5, > -0.5 to < +0.5, > +0.5). Minimization is a form of adaptive treatment allocation in which the 
probability of assignment to the treatment groups does not remain constant but is determined by the 
current balance and/or composition of groups. In this study, the treatment assignment probability will be 
adjusted in response to the marginal distributions of categories of age at entry and baseline executive 
function such that the marginal distributions of these categories will be approximately equal between 
treatment groups. Treatment group assignment will be generated and stored in a secured database.  
 
Data analysis: Preliminary Analyses. Exploratory analyses will be performed for data description and 
screening for anomalies (e.g., outliers, nonnormality). This will be done to: 1)describe univariate and 
bivariate distributions; 2) identify group imbalances and associations between dependent variables and 
suspected covariates/ confounders;3) evaluate missing data; and 4) check for violation of statistical 
assumptions. If assumptions are violated, data transformations or more statistically robust procedures will 
be considered. Covariates/ confounders will be included in models secondarily and their effects on 
primary predictors/factors will be evaluated. The randomness of missing data will be investigated using 
available information on subject characteristics to help discern patterns in missing data, identify possible 
missing data mechanisms and inform strategies to handle missing data. If the data are ignorable missing, 
the estimation procedures to be used will produce unbiased estimates while allowing us to retain 
observations with missing values on the outcome variables. If needed, multiple imputation would be used 
to impute missing values on covariates. If the data are not ignorable missing, we may use selection or 
pattern mixture modeling to investigate the sensitivity of results.  
 
Analysis Plan for Aim 1. An "intent-to-treat" (ITT) approach will be used for data analysis. All subjects will 
be included in the groups to which they were assigned, regardless of adherence to protocol, treatment 
received, withdrawal or protocol deviation. Although this approach is recommended for efficacy analyses 
in Retaste sensitivity of the results assuming ITT will be explored using information collected regarding 
participant’s adherence to the exercise intervention (e.g., protocol, amount of treatment received) ) as 
well as the amount of activity engaged in by the usual care group. The primary endpoint, changes in 
cognitive function, will be derived from normed(for age, education) composites of neuropsychological test 
scores (expressed as mean Z-scores).1 (Table 2) The composite scores for the targeted cognitive 
function domains are empirically derived via exploratory factor analysis. Given the variation in scaling, test 
scores are converted to Z-scores related to an established cohort of age- and education-matched healthy 
women, and then averaged into composites, where negative scores indicate poorer cognitive function. 
Linear mixed-effects modeling with linear contrasts will be used to examine the effect of treatment 
assignment on cognitive function over time. When fitting models, time will be repeated within-subjects 

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factor, while treatment assignment will be a between-subjects factor, with an interaction between time and 
group. Each derived cognitive domain composite will be modeled separately and interaction terms will be 
included to test our hypothesis that executive, memory, and attention domains will be more affected than 
other domains. We will consider baseline cognitive function values both as an assessment point and as a 
possible covariate in analyses. Fixed and/or time-dependent covariates may be included to adjust for 
group imbalances or variables related to the dependent variables. Standard fit indices will also be used to 
identify the most appropriate covariance structure. F-tests will test the main and interaction effects 
included in the model. Individual regression parameters will be computed and reported with standard 
errors to yield confidence intervals. For each model, residual analysis will be conducted to identify 
sources of model misspecification, outliers and influential observations. Sensitivity analyses will be 
performed to discern the impact of influential cases on results. To test specific hypotheses, linear 
contrasts will be specified and estimated in the repeated measures model to compare changes from 
follow-up to baseline values between groups. Wald t-statistics will be used to test each contrast. Marginal 
modeling with generalized estimating equations will also be used as it tends to be more robust to 
misspecification of the covariance structure and violations in normality assumptions. Results from each 
modeling approach will be compared via sensitivity analyses.  
 
Analysis Plan for Aim 2. We will use the same analytical approach described for Aim1 to fit models for 
intermediate outcomes (neuroimaging metrics of brain heath, inflammatory markers, and symptoms) and 
to test Aim 2 hypotheses regarding the effects of exercise on primary intermediate outcomes 
(neuroimaging metrics and inflammatory markers of IL-6 and CRP). The only difference will be that initial 
neuroimaging analyses will be conducted on a voxel-wise basis using FSL, SPM, treasurer, depending on 
the imaging modality and analysis (e.g., volumetric). In addition to voxel-wise analyses we may apply 
regions-of-interest (ROI) analyses since our primary hypotheses focus on prefrontal and hippocampal 
regions. Thesaurus can be generated using anatomical templates from normalized MNI space or from 
functionally-derived estimates of activation or connectivity. We will use standard cluster-based corrections 
for multiple comparisons of fMRI results, threshold-free cluster enhancement, and an FDR of p<.01 for 
volumetric and white matter analyses.  
 
Analysis Plan for Aim 3. We will explore neuroimaging metrics of brain health, biomarkers, and symptoms 
as variables that may mediate the effects of the exercise intervention on cognitive function and E2 as a 
possible moderator. Initially, we will fit simple mediational models (predictor, single mediator, single 
outcome) applying the change score method and estimate whether effects of exercise (predictor) on 
changes in cognitive function outcomes are mediated through changes in the suspected mediators. 
Goodness-of-fit will be assessed through standard summary indices (e.g., RMSEA, CFI) and residual 
analyses. Total, indirect and direct effects will be estimated and reported with confidence intervals. 
Additionally, we will explore mediation applying the half-longitudinal approach proposed by Cole 
andMaxwell_ENREF_148 as well as an autoregressive mediational approach. Depending on the results 
from simple mediational modeling, we may also combine mediators into a multiple or serial mediator 
models to develop a more comprehensive picture of these pathways. As stated previously, voxel-based 
mediation of neuroimaging results will be conducted using BRAVO by employing the same approach 
described above. To explore E2 as a moderator of exercise effects on cognitive function, the models 
developed in Aim 1 will be expanded to also include E2 as main effect as well as interactions of E2 with 
the other fixed effects. Exploratory analysis. We will explore whether physical activity diary data are 
related to objective physical activity data. If validated, we will explore the physical activity patterns over 6 
months and its relationship to change in outcomes.