A Feasibility Study of Tree Nut and Extra Virgin Olive Oil Supplementation to Improve Cardiometabolic Health
Phase: NA
Conditions: Metabolic Syndrome
Sponsor: Wake Forest University Health Sciences

 
 
 
 
 
A Feasibility Study Incorporating DNA Methylation-Predicted Aging with a  
Dietary Intervention to Improve Cardiometabolic Health 
 
1/14/2022 
 
IRB00065273 
 
NCT04361617 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
A Feasibility Study Incorporating DNA Methylation-Predicted Aging with a  
Dietary Intervention to Improve Cardiometabolic Health 
 
 
Principal Investigator: 
 
 
 
 
Co-Investigators: 
Lindsay M. Reynolds, PhD 
lireynol@wakehealth.edu 
336-713-1134  
 
 
Mara Z. Vitolins, DrPH, MPH, RDN  
mvitolin@wakehealth.edu 
 
Carl Langefeld, PhD  
clangefe@wakehealth.edu 
 
Funding Source:  
                                                              
Pilot award funded by Wake Forest Clinical and 
Translational Science Institute (CTSI) and Cardiovascular 
Sciences Center  
 
 
 
 
Date: 
June 5, 2020 
 
February 17, 2021 
 
March 24, 2021 
September 1, 2021 
November 5, 2021 
November 30, 2021 
 
December 21, 2021 
 
January 14, 2022 
 
 
 
 
 
 

 
3 
 
SCHEMA 
 
We will recruit up to 50 women and men (> 35 years) from the surrounding communities with 
Metabolic Syndrome (Mets). All participants will provide blood samples to determine 
epigenetic age estimates. Participants who meet eligibility criteria will be asked to participate 
in a 4-week nuts and extra virgin olive oil (EVOO) intervention. Half of the intervention 
participants will be randomly selected to be educated about epigenetic age acceleration before 
the intervention. All participants will have a phone visit after one week of the intervention to 
assess compliance, and all participants will be contacted by phone after week two of the 
intervention. Participants will have in person study-related measurements collected at baseline 
and 4 weeks.  
 
Self-referral (with health care provider assistance in identifying candidates) 
Telephone screening 
 
Baseline Visit: Informed consent and in-person assessments 
Blood pressure, weight, height, waist circumference, blood draw  
(other study measures as listed in section 5.3) 
 
Ineligible for intervention: 
Doesn’t meet MetS criteria  
Eligible for intervention: 
Meets MetS criteria  
 
Intervention visit for eligible participants 
EVOO and nuts intervention, with 1:1 random assignment to intervention arm educated on 
epigenetic age acceleration or intervention arm not educated on epigenetic age acceleration, 
blood draw 
 
                 EVOO and nuts intervention arm 1 
  EVOO and nuts intervention arm 2 
                           Educated on epigenetic aging   
       Not educated on epigenetic aging 
 
 
 
                 
 
Adherence assessment Telephone contact, end of week 1 
Assess adherence and safety, answer participant questions 
 
Telephone contact, end of week 2 
Assess adherence and safety, answer participant questions 
 
Final measurement visit, end of week 4 
Blood pressure, weight, height, waist circumference, blood draw  
 (other study measures as listed in section 5.3)  
Exit interview - Qualitative assessment of participant experience 
 

 
4 
 
Table of Contents 
SCHEMA ........................................................................................................................................................  
Table of Contents ............................................................................................................................................  
1.0 
Introduction and Background..............................................................................................................  
1.1 
Primary Objectives .................................................................................................................  
1.2 
Secondary Objectives .............................................................................................................  
1.3 
Exploratory Objectives ...........................................................................................................  
2.0 
Participant Selection ...........................................................................................................................  
2.1 
Inclusion Criteria ....................................................................................................................  
2.2 
Exclusion Criteria ...................................................................................................................  
2.3 
Inclusion of Women, Minorities and Children .......................................................................  
2.4 
Identification of Cohort ..........................................................................................................  
3.0 
Recruitment Procedures ......................................................................................................................  
3.1 
Epigenetic age acceleration ....................................................................................................  
4.0 
Treatment Plan: Tree Nuts and Extra Virgin Olive Oil ......................................................................  
4.1 
Toxicities ................................................................................................................................  
4.2 
Table of Study-Related Interventions .....................................................................................  
4.3 
Study Visits ............................................................................................................................  
4.3.1  
Basline Visit : In-person screening visit .................................................................................  
4.3.2  
Interventionvisit ......................................................................................................................  
4.3.3 
End of week 1 phone call ........................................................................................................  
4.3.4 
Final measurement visit, end of week 4 ..................................................................................  
5.0 
Study Measures ...................................................................................................................................  
5.1 
Anthropometric Measures .......................................................................................................  
5.2 
Blood Biomarkers ...................................................................................................................  
5.3 
Questionnaires and Other Measures........................................................................................  
5.4 
Duration of Participation ........................................................................................................  
5.5 
Duration of Follow Up ...........................................................................................................  
5.6 
Criteria for Removal from Study ............................................................................................  
6.0 
Adverse Events and Reporting Requirements.....................................................................................  
6.1  
WFHS IRB AE Reporting Requirements ...............................................................................  
7.0 
Data reporting  ....................................................................................................................................  
 

 
5 
 
8.0 
Statistical Considerations  ...................................................................................................................  
 
8.1 
Prevalence of epigenetic age acceleration  ..............................................................................  
8.2 
Assess adherence.....................................................................................................................  
8.3 
Participants’ perception of epigenetic age, challenges and motivators ...................................  
8.4 
Test for changes in epigenetic age acceleration ......................................................................  
8.5 
Test for changes in MetS-related traits ...................................................................................  
8.6 
Sample size justification .........................................................................................................  
 
References .......................................................................................................................................................  
Appendix A Consumption Diary ....................................................................................................................  
Appendix B  14-item Questionnaire of Mediterranean diet adherence ...........................................................  
Appendix C Intervention Experience Assessment  .........................................................................................  
Appendix D Perception of Epigenetic Age Assessment .................................................................................  
 
 
 
 
 
  
  
 
 
 
 
1.0 
Introduction and Background 
 
Metabolic syndrome (MetS) is a collection of at least three out of five cardiometabolic risk 
factors including abdominal obesity, hypertension, elevated blood glucose, hypertriglyceridemia, 
and low high-density lipoprotein cholesterol [1]. The prevalence of MetS is rising worldwide, 
and as of 2012, more than one third of all US adults met the criteria for MetS [2].  This is 
important as MetS is a major risk factor for cardiovascular disease and is considered a state of 
prediabetes [3]. Improving metabolic parameters through dietary, behavioral, and 
pharmacological interventions can improve or reverse MetS [4, 5]. For instance, a 5-year study 
in Spain (PREDIMED) evaluated the effects of the Mediterranean diet on primary prevention of 
cardiovascular disease in subjects at high risk of cardiovascular disease (CVD)[6].  PREDIMED 
was a multicenter, randomized, controlled clinical trial of 7,447 participants who were 
randomized to one of three diets: Mediterranean diet supplemented with extra virgin olive oil 
(EVOO), Mediterranean diet supplemented with mixed nuts, or a low-fat control diet. The 
PREDIMEDstudy found a reduction in the cardiovascular event rates in both intervention groups 
compared to the low fat control group. Additionally, post-hoc analysis of individuals meeting the 
criteria for MetS at baseline revealed EVOO and nut consumption (compared to controls) was 
more likely to result in reversion of MetS (HR = 1.35 95% CI: 1.15 – 1.58, p<0.001), and 
significant reductions in central obesity (p<0.001) and high fasting glucose (p = 0.02) compared 
to the low fat control group [7].  However, lower levels of adherence to the PREDIMED 
intervention was found in participants with a higher number of cardiovascular risk factors, larger 
waist circumference, and lower physical activity levels at baseline [8]. New strategies to convey 

 
6 
 
one’s risk of morbidity and mortality at the onset of a dietary intervention may improve 
intervention adherence, particularly among individuals meeting the criteria for MetS. 
DNA methylation patterns change with age, and can be used to estimate chronological age [9-
12]. A greater DNA methylation-based predicted age relative to chronological age, often referred 
to as “epigenetic age acceleration”, has been associated with many lifestyle factors, including 
physical activity and diet, as well as components of MetS, including obesity, blood pressure, 
HDL cholesterol and blood glucose levels [13]. A higher number of MetS components are cross-
sectionally associated with epigenetic age acceleration [14]. Epigenetic age acceleration is more 
strongly predictive for the risk of morbidity and mortality than chronological age [9, 11]. We 
hypothesize that the majority of people with MetS have advanced epigenetic aging, and among 
those that have advanced epigenetic age, learning about epigenetic age and its relationship to 
morbidity and mortality at the onset of a dietary intervention will improve participant adherence 
to the dietary intervention. Further, we hypothesize that daily consumption of EVOO and tree 
nuts in participants with MetS and advanced epigenetic age could help reverse MetS and 
potentially slow epigenetic aging.  Therefore, we plan to conduct this feasibility pilot study to: 
 
1.1 
Primary Objectives 
To determine the proportion of participants with MetS with epigenetic age 
acceleration 
To assess adherence to daily EVOO and tree nut consumption over the 4-week 
intervention, and compare adherence in participants educated about epigenetic age 
acceleration at baseline compared to intervention arm not educated about 
epigenetic age acceleration 
Qualitatively explore how participants perceive epigenetic age, and how 
participants’ experiences would impact the feasibility of a larger clinical 
intervention in terms of challenges and motivators  
1.2 
Secondary Objectives 
Compare epigenetic age acceleration before and after the 4-week intervention 
 
1.3 
Exploratory Objectives 
Compare changes in MetS related traits (body mass index, waist circumference, 
blood pressure, HDL cholesterol, triglycerides, HbA1c, and fasting glucose 
levels) before vs. after the 4-week intervention  
2.0 
Participant Selection 
This study can fulfill its objective only if participants appropriate for this intervention are 
enrolled. All relevant medical and other considerations will be taken into account when 
deciding whether this protocol is appropriate for a particular candidate.  
 
2.1  
Inclusion Criteria  
2.1.1 Men and Women ≥ 35 years of age 
2.1.2 Metabolic syndrome, defined as > 3 of the following:   

 
7 
 
2.1.2.1  Waist circumference >102 cm in men and >88cm in women 
2.1.2.2  Triglycerides >150 mg/dL AND/OR drug treatment for elevated 
triglycerides 
2.1.2.3  HDL cholesterol <40 mg/dL in men and <50 mg/dL in women 
AND/OR drug treatment for reduced HDL cholesterol 
2.1.2.4  Systolic blood pressure >130 mm Hg or diastolic blood pressure 
>85 mmHg AND/OR antihypertensive drug treatment 
2.1.2.5  Fasting glucose >100 mg/dL or hemoglobin A1c > 5.6% AND/OR 
oral hypoglycemic medications 
2.1.3 Willing to comply with study visits, as outlined in the protocol 
2.1.4 Able to read and speak English 
2.1.5 No allergies or hypersensitivities to olive oil or nuts   
2.1.6 Ability to understand and the willingness to sign a written informed consent 
document. 
2.1.7 Completion of a validated 14-item Mediterranean Diet Assessment Screener to 
assess baseline dietary consumption of nuts and olive oil 
2.2 
Exclusion Criteria  
2.2.1 Plans to move from the study area in the next 12 weeks 
2.2.2 Body Mass Index (BMI) > 40 kg/m2 
2.2.3 Dementia that is medically documented or suspected, or clinical evidence of 
cognitive impairment sufficient to impair protocol adherence 
2.2.4 Candidate with any dietary practice, behavior or attitude that would substantially 
limit ability to adhere to protocol 
2.2.5 Homebound for medical reasons 
2.2.6 Living in the same household with another participant 
2.2.7 Insulin-dependent Diabetes 
2.3 
Inclusion of Women, Minorities and Children 
Males and females of all races and ethnic groups are eligible for this trial. Children will not be 
included in this study as children with metabolic syndrome may have very different underlying 
etiologies for metabolic syndrome than adults. 
 
2.4  
Identification of Cohort 
We propose to draw our sample of 50 men and women from the population residing in Winston-
Salem/Forsyth County, North Carolina.  Over 361,220 persons live in the county.  In addition, 
over 1.3 million people reside in the eight contiguous counties comprising the Piedmont Triad 
area.  We anticipate that recruitment for the study can be accomplished in Forsyth County.  
However, if needed, we will extend recruitment to the surrounding communities. 
 
3.0 Recruitment Procedures 
Investigators at Wake Forest School of Medicine have extensive experience in recruitment for 
both observational and clinical trials.  Based on our previous experience in large-scale studies, 
we have found the use of several approaches for recruitment substantially better than relying on 
one recruitment strategy.  These approaches include: community-wide methods designed to 
reach large numbers of men and women and contacting participants through MyWakeHealth 
communications.  We will also recruit participants through advertising on the WFBH website 

 
8 
 
‘Be Involved’ and using study flyers posted at local medical clinics including Family Medicine 
and Internal Medicine. 
 
We anticipate it will take approximately 12 weeks to recruit 50 participants for this project. 
Candidates will complete a telephone screening.  The baseline visit will consist of an in-person 
assessment visit, scheduled for a morning time, and participants will be asked to be fasting and 
will be instructed to bring their medications to the visit. Candidates will review the informed 
consent document at the baseline visit for in-person screening with study staff prior to beginning 
their participation. 
 
Each candidate will be greeted by a study staff member who will take the candidate to a private 
consulting room.  A final eligibility checklist and each section of the informed consent will be 
reviewed with the candidate.  Once the candidate fully understands each section of the consent, 
he/she will sign the document and be given a copy to take home. A copy will be retained by the 
study staff.  
 
Recruitment of Participants 
 
The goal of the recruitment phase is to enroll 50 men and women with MetS into the study.   
 
 
 
 
 
 
 
 
Female 
Male 
American Indian or Alaska Native   
 
 
    1 
 
   1 
Asian or Pacific Islander 
 
 
 
 
    1 
 
   1 
Black, not of Hispanic Origin  
 
 
 
    6 
 
   6 
Hispanic 
 
 
 
 
 
 
    2 
 
   2 
White, not of Hispanic Origin 
 
 
 
   14 
              14 
Other or Unknown 
 
 
 
 
 
    1 
 
   0 
TOTAL                                                                                      25  
  25 
 
3.1 
Epigenetic Age Acceleration 
For all participants providing informed consent, blood samples will be collected at each in-
person visit to calculate epigenetic age acceleration.  
 
4.0 
Treatment Plan: Tree Nuts and Extra Virgin Olive Oil  
At the intervention visit, all participants will receive the full 4-week supply of EVOO and tree 
nuts, including unsalted English walnuts, almonds or pistachios (approximately 10-day supply of 
each type). Participants will be asked to supplement their normal diets with these products and 
will be provided recipes and other information that will allow them to replace other foods with 
the nuts and oil. We will ask participants to consume one ounce of tree nuts per day and two 
tablespoons of EVOO per day by incorporating these foods into their diet. Dietary adherence 
diaries will be given to measure incorporation of the study foods.  
 

 
9 
 
Participants will be randomized to learn about epigenetic age acceleration (arm 1) or not to learn 
about epigenetic age acceleration (arm 2) in a 1:1 allocation at the intervention visit.  Those in 
the intervention arm 1 that are to learn about epigenetic age acceleration will receive some 
educational materials and a brief description of epigenetic age acceleration.  
 
A telephone visit will be conducted at the end of week 1 for additional diet counseling, to assess 
safety/potential side effects, to collect adherence diaries. A follow-up phone call for compliance 
assessment and to answer participant questions will be conducted at the end of week 2. A final 
measurement visit will be scheduled for the end of week 4 for a morning time and participants 
will be asked to be fasting. 
 
4.1 
Toxicities 
Side effects will be monitored by staff contact at the end of week 1 visit, and at the end of week 
2 (by phone) and at the final measurement visit at the end of week 4.  Participants will be 
encouraged to contact the Study manager during the study period if they feel they are 
experiencing effects related to the study foods. 
 
EVOO and tree nuts are generally recognized as safe and are consumed by free living adults on a 
regular basis.  The amount to be given in this study are within the range given to the participants 
in the PREDIMED study [6] and found to have only minor side effects, mainly stomach upset, 
bloating and nausea, and none of the side effects advanced to serious adverse events.  
 
Administration 
Study foods will consist of one ounce of tree nuts per day and two tablespoons of EVOO per 
day.  Participants will be instructed how to consume the proper amount of tree nuts and EVOO 
and reporting the consumption on the compliance diary (Appendix A). The tree nuts will be 
provided in daily servings (one ounce per day) and EVOO will be provided in bottles, along with 
a tablespoon. Participants will be instructed to use the tablespoon to measure EVOO.  
Participants will also receive recipes and other written information that will assist them in 
incorporating both the nuts and EVOO into their existing dietary patterns.  
 
Dose Modification/Toxicity management:   
Side effects will be monitored via the telephone visit at the end of week 1 and 2 and by 
participant report. Participants will be encouraged to contact the study team during the study 
period if they feel they are experiencing side effects related to the study foods.  For those 
participants with existing medical conditions (e.g., gallbladder removal or bariatric surgery) or 
who are experiencing nausea related to consumption of the olive oil, a titrated dosing plan will 
be used. These participants will begin their olive oil consumption at 1 tablespoon/day for 5 days 
and increasing to 2 tablespoons/day on the sixth day if it is well tolerated. If this increase is not 
tolerated, the participant can be returned to 1 tablespoon per day and attempt an increase after an 
additional 5 days.  
 
 
 
Withdrawal from study intervention 

 
10 
 
Any side effects experienced by the participant may or may not be due to the study foods. If the 
side effects become intolerant for the participant, he/she may be withdrawn from the study at the 
discretion of the study team. 
4.2 
Table of Study-Related Interventions  
 
Measure 
Baseline: 
In-person 
screening 
visit 
 Intervention  
visit 
End of 
Week 
1 
Phone 
Call 
End of 
week 2 
Phone 
Call 
Final 
in 
person 
visit 
Anthropometrics 
Body weight 
Height 
Waist circumference 
 
X 
X 
X 
 
 
 
 
X 
X 
X 
Blood collection 
X 
X 
 
 
X 
Blood pressure 
X 
 
 
 
X 
Demographics 
X 
 
 
 
 
Mediterranean diet screener 
X 
 
 
 
X 
Health History/health habits 
X 
 
 
 
X 
Adherence/safety assessment 
 
 
X 
X 
X 
Intervention Experience Assessment 
and Perception of Epigenetic Age 
Assessment (arm 1 only) 
 
 
 
 
X 
Dispense study foods 
 
X 
 
 
 
 
4.3 
Study Visits 
4.3.1  
Baseline Visit: In-person screening  
At the in-person screening visit each participant will be greeted by a staff member who will take 
the candidate to a private consulting room.  A final eligibility checklist and each section of the 
informed consent will be reviewed with the candidate.  Once the candidate fully understands 
each section of the consent, he/she will sign the document and be given a copy to take home. A 
copy will be retained by the study staff.  
 
After providing informed consent, fasting participants will complete study questionnaires in a 
quiet private room and show current medications to a staff member that will record the names of 
medications brought to the clinic. Next, blood pressure will be measured after resting 5 minutes 
and sitting upright.  Blood pressure may be measured twice.  Body weight will then be measured 
using a beam scale with movable weights with participant wearing indoor clothing with no 
shoes.  Height and waist circumference will also be measured. Participant will also have a fasting 
blood collection. Participants will be asked to complete a validated 14-item Questionnaire of 
Mediterranean diet adherence [15] to assess their baseline diet including consumption of olive oil 
and nuts (Appendix B). 
4.3.2 
Intervention visit 
Eligible participants (those that provided informed consent, were fasting at the in-person visit 
and met the criteria for MetS, and provided blood samples for epigenetic age acceleration 

 
11 
 
measurements) will be contacted to schedule the intervention visit. The study is set to end on 
March 21, 2022, which means the study intervention visits need to be completed by March 3, 
2022. Participants that have not attended their intervention visit by March 3, 2022 will be 
discontinued from the study. 
 
At the intervention visit, participants will have a fasting blood collection. Participants will 
receive counseling and recipes to help them incorporate the nuts and EVOO into daily meals. 
Dietary adherence diaries will be given to participants to measure incorporation of the study 
foods and participants will be instructed to complete the adherence diaries on a daily basis. 
Participants will be given a 4-week supply of EVOO, tree nuts, and a tablespoon for measuring 
the EVOO.  
 
Participants will be randomized to learn about epigenetic age acceleration (arm 1) or to not learn 
about epigenetic age acceleration (arm 2) in a 1:1 allocation at the baseline visit.  Those in arm 1 
will receive some educational materials and a brief explanation of epigenetic age acceleration. At 
the end of this visit, all participants will be scheduled for the remainder of their telephone and in 
person study visits. 
4.3.3 
End of week 1 and week 2 telephone calls 
During the end of week 1 telephone call, participants will be contacted via telephone and asked 
questions about their adherence, and any potential side effects observed. Participants will receive 
additional counseling for how to incorporate the nuts and oil into a daily meal as needed. A 
follow up phone call at the end of week 2 will be scheduled to assess compliance and to answer 
participant questions, and a final measurement visit will be scheduled for the end of week 4 for a 
morning time and participants will be asked to be fasting. 
4.3.4 
Final measurement visit, end of week 4 
The same measures, as listed in the in-person screening visit will be performed.  Study 
questionnaires, with the exception of demographics and health history, will be distributed and 
completed, and adherence diaries will be collected.  
For participants of the intervention arm 1 (participants educated about epigenetic age 
acceleration), a self-administered exit questionnaire will be given to qualitatively explore 
participants’ perception of epigenetic age, and challenges and motivators for behavior change 
during the intervention. The open-ended questions will be designed to ascertain understanding of 
epigenetic age, and assess the challenges and motivators participants encountered during the 
intervention.  
Participants in both arms will be administered an Intervention Experience Assessment to explore 
how participants’ experiences would impact the feasibility of a larger clinical intervention in 
terms of challenges and motivators. 
 
5.0 
Study Measures 
5.1 
Anthropometric Measures 
Weight: Weight will be assessed using a digital scale in kilograms with participants wearing 
indoor clothing with no shoes.   

 
12 
 
 
Height: Height will be measured using a stadiometer attached to a beam scale, in centimeters, 
with no shoes worn.   
 
Blood pressure: First-phase systolic and fifth-phase diastolic Korotkoff blood pressure will be 
auscultated from the right arm of the subject, after resting 5 minutes and sitting upright.  We will 
use a standard manometer with a cuff sized appropriately.  .  If the blood pressure reading is 
elevated, it will trigger an "alert" and subsequent participant/physician notification.   
 
Waist circumference: Waist circumference will be measured at the midpoint between the lowest 
rib and the iliac crest.  
 
5.2  
Blood-based Biomarkers 
 
HDL cholesterol, triglycerides, HbA1c, and fasting glucose levels will be analyzed using serum 
samples at a local LabCorp facility. 
 
Epigenetic age acceleration: DNA methylation will be measured from blood samples frozen/ 
stored at -80 C within 2 hours of collection. After recruitment of 50 participants meeting the 
criteria for MetS is completed, blood samples will be sent for DNA methylation analysis in the 
lab of Dr. Howard at WFBMC. DNA will be extracted from whole blood samples, and will be 
bisulfite-converted using the EZ DNA Methylation Gold kit (Zymo, Irvine, CA). Illumina 
Infinium MethylationEPIC BeadChip, which targets over 850,000 CpG sites, and the Illumina 
iScan Reader will be used to determine the proportion of DNA methylation at each site.  The 
ratio of the fluorescence intensity of the methylated versus the combined methylated and 
unmethylated probes will be determined with Illumina GenomeStudio. As a quality control 
measure, the cumulative fluorescent signal must be significantly greater than the negative 
controls included for each sample. We will use a threshold of this “detection p-value” of ≤0.05 in 
at least 90% of the samples as criteria for adequate assays. Finally, adjustments will be made to 
normalize the fluorescent signals from the Infinium I and Infinium II fluorescent assays used 
prior to analysis (in Bioconductor).  
DNA methylation profiles (beta-value, after adjustment chip and position effects) at 1,030 
unique DNA methylation profiles will be used for epigenetic age acceleration predictions, using 
the DNA GrimAge predictor [11]. We will calculate predicted lifespan and regress predicted 
lifespan on chronological age to produce estimates of epigenetic age acceleration.  
5.3 
Questionnaires 
Demographic information: Name, marital status, date of birth; racial/ethnic group; occupation; 
education. 
 
Personal medical history: Select questions about the participant’s medical history, past and 
present. 
 
Dietary intake: Participants will be asked to complete Mediterranean Diet 14 item dietary 
screener.  
 

 
13 
 
Current medications: Prescription medications; participants will be instructed to bring 
medications to the clinic.  
 
Intervention Assessments (see appendix A) 
Compliance-Intake of the study foods, tree nuts and EVOO will be tracked on a daily basis using 
a compliance diary.  The participant will record on a daily diary the following information: 
Did you eat the nuts today? - Yes, No 
Did you consume the oil today? -Yes, No 
 
Telephone contacts and maintenance of the cohort:  Telephone contact will be used to monitor 
participants for study compliance and side effects by phone at the end of week 1 and 2.  During 
the phone call participants will be invited to ask any questions and to discuss any problems with 
the study, specifically their adherence to the study foods.  Participants’ medication history will 
be reviewed.  
 
 Perception of Epigenetic Age Assessment -For participants of the intervention arm 1 
(participants educated about epigenetic age acceleration), a self-administered exit questionnaire 
will be given to qualitatively explore participants’ perception of epigenetic age, and challenges 
and motivators for behavior change during the intervention. The questions will be designed to 
ascertain understanding of epigenetic age, and assess the challenges and motivators participants 
encountered during the intervention.  
 
Intervention Experience Assessment- Participants in both study arms will be administered an 
intervention experience assessment to explore how participants’ experiences would impact the 
feasibility of a larger clinical intervention in terms of challenges and motivators. 
 
5.4 
Duration of Participation in Intervention 
Participation will be for a 4-week period unless the following occur:  
• 
Illness that prevents further administration of treatment 
• 
Unacceptable adverse event(s) 
• 
Patient decides to withdraw from the study 
• 
General or specific changes in the participant’s condition render the patient unacceptable 
for further treatment in the judgment of the investigator 
5.5 
Duration of Follow Up 
Participants will be involved in the study intervention for a period of 4 weeks. Participants 
removed from study for unacceptable adverse events will be followed until resolution or 
stabilization of the adverse event.  
5.6 
Criteria for Removal from Study 
Participants will be removed from study when any of the criteria listed above applies.  The 
reason for study removal and the date the participant was removed will be documented in the 
Case Report Form. 
The study is set to end on March 31, 2022. As such, all study intervention visits need to be 
completed by March 3, 2022. Any participants remaining on study, that have not attended their 
intervention visit by that date, will be discontinued from the study. 
 

 
14 
 
6.0 
Adverse Events and Reporting Requirements 
The risk of harm or discomfort that may happen as a result of taking part in this research study is 
not expected to be more than in daily life or from routine physical or psychological examinations 
or tests.    
 
There is risk, although minor, involved in obtaining blood samples.  There may be some 
discomfort, bruising and/or bleeding where the needle is inserted for blood draws.   Some people 
become dizzy, lightheaded, or feel faint.  Infection may occur on rare occasions.  Frequent 
donation of blood can result in low iron in the blood.   
 
There is also a risk of weight gain from the increased caloric intake that may occur as a result of 
consuming the nuts and oils.  Participants will be provided with recipes and other information on 
incorporating the nuts and oils by replacing other foods to reduce the risk of weight gain and will 
be encouraged to contact the study team if they believe they are gaining weight.   
 
There is a slight risk that study participants may have an unknown allergy to nuts or olive oil and 
may therefore have an allergic reaction to one of these substances. Mild allergic symptoms that 
can occur include: raised red bumps of skin – hives, swelling of the lips, tingling of the throat 
and mouth, itchy skin and rash, runny nose, tightening of the throat, and digestive symptoms – 
cramps, stomach pain, nausea, or vomiting. If study participants feel they are experiencing one of 
these mild allergic reactions, they will be instructed to contact the study team and report the 
reaction. 
 
Although unexpected in adults without prior knowledge of an allergy to tree nuts, consumption 
of tree nuts can also result in severe allergic reaction symptoms including: difficult or noisy 
breathing, swelling of the tongue, swelling or tightness of the throat, difficulty talking or a hoarse 
voice, wheeze or persistent cough, or persistent dizziness or collapse. If study participants feel 
they are experiencing one of these severe allergic reaction symptoms, they will be instructed to 
call 911 and seek immediate medical care. After treatment, they will be instructed to contact the 
study team and report the reaction. 
 
Study staff will report any adverse events to the PI, Dr. Reynolds, who will review all 
unanticipated problems involving risk to participants or others, serious adverse events, and 
provide a written report of the event to the IRB.  If necessary, the study team will direct 
appropriate referrals for health care needed to address study-related adverse events.  
 
Results of routine clinical labs and physical measures obtained as part of study visits that meet the 
criteria for “Alert Values” will be communicated as described in the table below.  The PI, Dr. 
Lindsay Reynolds, will review medical eligibility criteria as needed, as well as clinical measures 
and laboratory reports. Dr. Kristina Lewis, the Study Medical Director, will serve as the primary 
contact for staff and participants regarding medical issues and labs and physical measures meeting 
alert values. Dr. Lewis will review summaries of alerts on a periodic basis along with the AEs.   
Alert Values 
 

 
15 
 
Measure 
Alert Value 
Notify Participant 
Notify PCP/Medical 
Director 
Blood 
Pressure 
(Avg.) 
Level 1 
SBP ≥180 
mm/Hg  OR 
DBP ≥110 mm/Hg 
In clinic. Advise to 
follow-up with PCP 
within 1 week. 
Within 1 week. 
IF symptomatic (e.g. chest 
pain, headache, short of 
breath), notify safety officer 
and/or PCP immediately. 
Level 2 
SBP ≥160 
mm/Hg  OR 
DBP ≥100 mm/Hg 
(and not Level 1 BP) 
In clinic. Advise to 
follow-up with PCP 
within 1 month. 
Within 1 week.   
IF symptomatic (e.g. chest 
pain, headache, short of 
breath), notify safety officer 
and/or PCP immediately. 
Level 3 
SBP ≥140 
mm/Hg  OR 
DBP ≥90 mm/Hg 
(and not Levels 1 or 
2) 
In clinic. Advise to 
follow-up with PCP 
within 2 months. 
Per routine reporting. 
IF symptomatic (e.g. chest 
pain, headache, short of 
breath), notify safety officer 
and/or PCP immediately. 
SBP ≤90 
mm/Hg  OR 
DBP ≤50 mm/Hg 
In clinic. Advise to 
follow-up with PCP 
within 1 week. 
Within 1 week.   
IF symptomatic (lightheaded, 
feels faint), notify safety 
officer and/or PCP 
immediately. 
Blood 
glucose 
< 50 mg/dl     OR 
> 400 mg/dl 
Within 1 week or sooner 
as indicated by exact 
value and clinician 
judgment. 
Within 1 week or sooner as 
indicated by exact value and 
clinician judgment. 
Triglycerides TG ≥ 1000 mg/dl 
Within 1 week or sooner 
as indicated by exact 
value and clinician 
judgment. 
Within 1 week or sooner as 
indicated by exact value and 
clinician judgment. 
 
 
 
6.1  
WFHS IRB AE Reporting Requirements 
Any unanticipated problems involving risks to subjects or others and adverse events shall be 
promptly reported to the IRB, according to institutional policy.  Reporting to the IRB is required 
regardless of the funding source, study sponsor, or whether the event involves an investigational 
or marketed drug, biologic or device.  Reportable events are not limited to physical injury, but 
include psychological, economic and social harm.  Reportable events may arise as a result of 
drugs, biological agents, devices, procedures or other interventions, or as a result of 
questionnaires, surveys, observations or other interactions with research subjects. 
 
All members of the research team are responsible for the appropriate reporting to the IRB and 
other applicable parties of unanticipated problems involving risk to subjects or others.  The 

 
16 
 
Principal Investigator, however, is ultimately responsible for ensuring the prompt reporting of 
unanticipated problems involving risk to subjects or others to the IRB.  The Principal 
Investigator is also responsible for ensuring that all reported unanticipated risks to subjects and 
others which they receive are reviewed to determine whether the report represents a change in 
the risks and/or benefits to study participants, and whether any changes in the informed consent, 
protocol or other study-related documents are required. 
 
Any unanticipated problems involving risks to subjects or others occurring at a site where the 
study has been approved by the WFHS IRB (internal events) must be reported to the WFHS IRB 
within 7 calendar days of the investigator or other members of the study team becoming aware of 
the event. 
 
7.0 
Data Reporting 
Data will be tracked and entered by study staff on a secure server.  No data reporting is 
necessary. 
 
8.0 
Statistical Considerations 
Objectives: This feasibility study, incorporating epigenetic age estimates with a nuts and EVOO 
intervention in individuals with MetS, is an initial step in building our understanding of the 
relationship between metabolic syndrome and epigenetic age and how epigenetic age estimates 
may be perceived by participants with MetS. This study will provide preliminary longitudinal 
data examining the potential to slow epigenetic age acceleration after a 4-week nuts and EVOO 
intervention. This longitudinal study is designed to provide pilot information that will be used to 
design a larger study of the effects of the nuts and EVOO on epigenetic aging.   This line of work 
will be an important foundation towards translating epigenetic age prediction from a research 
concept towards a tool to meaningfully promote healthy lifestyle changes to improve 
cardiometabolic health among individuals at high risk for cardiovascular disease.  
8.1  
Prevalence of epigenetic age acceleration 
Primary Outcome 1: proportion of participants with MetS with advanced epigenetic aging  
 
To characterize the relationship between MetS and epigenetic aging, we will examine epigenetic 
age acceleration prevalence among the 50 participants with metabolic syndrome. Epigenetic age 
acceleration will be calculated as described in 5.2. Advanced epigenetic age acceleration will be 
defined as a positive epigenetic age acceleration (higher than expected based on chronological 
age). 
8.2  
Assess adherence 
Primary Outcome 2: proportion of days for which EVOO was taken  
Primary Outcome 3: proportion of days for which tree nuts were taken 
Primary Outcome 4: proportion of days for which EVOO and tree nuts were taken 
 
For adherence, we will examine the daily diaries for intake of the two study foods, tree nuts and 
EVOO.  Adherence for each participant will be measured as the proportion of days in 4 weeks 
for which nuts, and of EVOO were taken, and by the proportion of days for which both were 
taken.  Problems associated with consuming the study foods will be defined as non-adherence 
(ceasing to consume either or both study foods) and low adherence (not consuming both foods 

 
17 
 
on a daily basis). The number of days on average each food was consumed and the overall 
completion rate of adherence diaries will be used to refine plans to both monitor and boost 
adherence in a future longer, larger trial.  
8.3  
Participants’ perception of epigenetic age, challenges and motivators 
Primary outcome 5: Perception of Epigenetic Aging Assessment and Intervention Experience 
Assessment 
  
To gain preliminary data supporting the feasibility of testing epigenetic age acceleration as a 
motivating factor for behavior change in a larger trial, participants of the intervention arm 1 
(participants educated about epigenetic age acceleration), will be given a self-administered exit 
questionnaire to qualitatively explore participants’ perception of epigenetic age.  All participants 
will given a self-administered Intervention Experience Assessment to explore participant 
experiences in the study, and the challenges and motivators for behavior change during the 
intervention. The \ questions will be designed to ascertain understanding of epigenetic age, and 
assess the challenges and motivators participants encountered during the intervention. All 
participants will be asked if they can be contacted for future studies. 
8.4  
Test for changes in epigenetic age acceleration 
Secondary outcome: Changes in epigenetic age acceleration 
 
It is not clear if changes in nut and EVOO consumption can slow or reverse epigenetic age 
acceleration. As a secondary goal, we will compare change in epigenetic age acceleration from 
the baseline visit to the intervention visit (control period) to change in epigenetic age 
acceleration from the intervention visit to the final visit  using a paired sample t-test.  
8.5 
Test for changes in MetS-related traits 
Exploratory outcomes: Changes in body mass index, waist circumference, blood pressure, HDL 
cholesterol, triglycerides, HbA1c, and fasting glucose levels  
 
Although longer clinical trials involving daily consumption of EVOO and nuts have observed 
improvement in MetS-related traits, it is unclear if improvements in MetS-related traits will be 
observed after 4-weeks. As an exploratory analysis, we will test for changes in MetS-related 
traits before vs. after the 4-week intervention, including changes in body mass index, waist 
circumference, HDL cholesterol, triglycerides, HbA1c, and fasting glucose levels using a paired 
sample t-test. 
8.6 
Sample size justification 
The prevalence of epigenetic age acceleration in people with metabolic syndrome is unknown. 
Also, it is unknown if a short term nuts and EVOO intervention will have measureable effects on 
estimates of epigenetic age. Thus, this pilot study will provide feasibility data necessary to 
estimate sample sizes for larger trails. Assuming 50 individuals and a type 1 error rate of α=0.05, 
the study has 0.80 power to detect difference of 0.4 standard deviations in the difference in 
epigenetic age and chronological age. We acknowledge that power is a limitation of the current 
study, and designed the study to maximize the sample size based on budget constraints to 
provide prevalence and potential effect size estimates to guide power calculations when planning 
a future larger, longer trial. 
 

 
18 
 
 
References 
 
1. 
Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC, 
James WP, Loria CM, Smith SC, Jr.: Harmonizing the metabolic syndrome: a joint 
interim statement of the International Diabetes Federation Task Force on 
Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American 
Heart Association; World Heart Federation; International Atherosclerosis Society; 
and International Association for the Study of Obesity. Circulation 2009, 120:1640-
1645. 
2. 
Moore JX, Chaudhary N, Akinyemiju T: Metabolic Syndrome Prevalence by 
Race/Ethnicity and Sex in the United States, National Health and Nutrition 
Examination Survey, 1988-2012. Prev Chronic Dis 2017, 14:E24. 
3. 
Grundy SM: Pre-diabetes, metabolic syndrome, and cardiovascular risk. J Am Coll 
Cardiol 2012, 59:635-643. 
4. 
Powell LH, Appelhans BM, Ventrelle J, Karavolos K, March ML, Ong JC, Fitzpatrick 
SL, Normand P, Dawar R, Kazlauskaite R: Development of a lifestyle intervention for 
the metabolic syndrome: Discovery through proof-of-concept. Health Psychol 2018, 
37:929-939. 
5. 
Grundy SM: Metabolic syndrome update. Trends Cardiovasc Med 2016, 26:364-373. 
6. 
Estruch R, Ros E, Salas-Salvado J, Covas MI, Corella D, Aros F, Gomez-Gracia E, Ruiz-
Gutierrez V, Fiol M, Lapetra J, et al: Primary Prevention of Cardiovascular Disease 
with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or Nuts. N 
Engl J Med 2018, 378:e34. 
7. 
Babio N, Toledo E, Estruch R, Ros E, Martinez-Gonzalez MA, Castaner O, Bullo M, 
Corella D, Aros F, Gomez-Gracia E, et al: Mediterranean diets and metabolic 
syndrome status in the PREDIMED randomized trial. Cmaj 2014, 186:E649-657. 
8. 
Downer MK, Gea A, Stampfer M, Sanchez-Tainta A, Corella D, Salas-Salvado J, Ros E, 
Estruch R, Fito M, Gomez-Gracia E, et al: Predictors of short- and long-term 
adherence with a Mediterranean-type diet intervention: the PREDIMED 
randomized trial. Int J Behav Nutr Phys Act 2016, 13:67. 
9. 
Chen BH, Marioni RE, Colicino E, Peters MJ, Ward-Caviness CK, Tsai PC, Roetker NS, 
Just AC, Demerath EW, Guan W, et al: DNA methylation-based measures of 
biological age: meta-analysis predicting time to death. Aging (Albany NY) 2016, 
8:1844-1865. 
10. 
Horvath S: DNA methylation age of human tissues and cell types. Genome Biol 2013, 
14:R115. 
11. 
Lu AT, Quach A, Wilson JG, Reiner AP, Aviv A, Raj K, Hou L, Baccarelli AA, Li Y, 
Stewart JD, et al: DNA methylation GrimAge strongly predicts lifespan and 
healthspan. Aging (Albany NY) 2019, 11:303-327. 
12. 
Hannum G, Guinney J, Zhao L, Zhang L, Hughes G, Sadda S, Klotzle B, Bibikova M, 
Fan JB, Gao Y, et al: Genome-wide methylation profiles reveal quantitative views of 
human aging rates. Mol Cell 2013, 49:359-367. 
13. 
Quach A, Levine ME, Tanaka T, Lu AT, Chen BH, Ferrucci L, Ritz B, Bandinelli S, 
Neuhouser ML, Beasley JM, et al: Epigenetic clock analysis of diet, exercise, 
education, and lifestyle factors. Aging (Albany NY) 2017, 9:419-446. 

 
19 
 
14. 
Nannini DR, Joyce BT, Zheng Y, Gao T, Liu L, Yoon G, Huan T, Ma J, Jacobs DR, Jr., 
Wilkins JT, et al: Epigenetic age acceleration and metabolic syndrome in the 
coronary artery risk development in young adults study. Clin Epigenetics 2019, 
11:160. 
15. 
Martinez-Gonzalez MA, Garcia-Arellano A, Toledo E, Salas-Salvado J, Buil-Cosiales P, 
Corella D, Covas MI, Schroder H, Aros F, Gomez-Gracia E, et al: A 14-item 
Mediterranean diet assessment tool and obesity indexes among high-risk subjects: 
the PREDIMED trial. PLoS One 2012, 7:e43134. 
 
 

 
20 
 
Appendix A 
 
 
 
 
 
 
 
Did you 
consume a 
one ounce 
portion of 
nuts today? 
Did you 
consume 2 
tablespoons 
of olive oil 
today? 

 
21 
 
Appendix B. 
 
14-item Questionnaire of Mediterranean diet adherence 
 
1. 
Do you use olive oil as your main cooking fat? 
1
 Yes 
 
 
2
 No 
 
2. 
How much olive oil do you consume in a given day (including oil used for frying, salads, restaurant 
meals, etc.)? 
tablespoons 
 
3. 
How many vegetable servings do you consume per day? (1 serving is ½ a cup of cooked 
vegetables or 1 cup of raw vegetables)  
servings 
 
4. 
How many fruit servings, including natural fruit juices, do you consume per day? ( 1 serving is a 
medium fresh fruit; ½ cup of chopped, cooked, or canned fruit; ½ cup of juice) 
 
servings 
 
5. 
How many servings of red meat, hamburger, or meat products (ham, sausage, etc.) do you consume 
per day? (1 serving is 2-3 ounces) 
 
servings 
 
6. 
How many servings of butter, margarine, or cream do you eat per day? (1 serving is 1 Tbsp.) 
 
servings 
 
7. 
How many sweet or carbonated beverages do you drink per day? 
 
beverages 
 
 
8. 
How much wine do you drink per week? 
 
glasses 

 
22 
 
 
9. 
How many servings of legumes do you consume per week? (1 serving  is ½ cup of beans or lentils) 
 
servings 
 
10. 
How many serving of fish or shellfish do you consume per week? (1 serving is 2-3 ounces) 
 
servings 
 
11. 
How many times per week do you consume commercial sweets or pastries (not homemade), such as 
cakes, cookies, pies, or pudding?  
 
times 
 
12. 
How many servings of nuts (including peanuts) do you consume per week? (1 serving is ¼ cup of nuts 
or 2 tablespoons of nut butter) 
 
servings 
 
13. 
Do you preferentially consume chicken, turkey, or rabbit meat instead of veal, pork, hamburger, or 
sausage?  
1
 Yes 
 
 
2
 No 
 
14. 
How many times per week do you consume vegetables, pasta, rice, or other dishes seasoned with 
sauce made with tomato and onion, leek, or garlic and simmered with olive oil? 
 
times 
 
 
 
 
 
 
 
 
 

 
23 
 
Appendix C 
 
STUDY EVALUATION  
 
 
 
 
 
1. How often during the course of your participation in this study did you replace foods in your 
diet with the tree nuts and olive oil?  
 
    -2 
 
       -1 
          0 
        1 
 
     2 
 
  Never 
 
Very Rarely 
 Occasionally 
 Very Often 
 
 Always 
 
 
 
2. How often during the course of your participation in this study did you eat the tree nuts and 
olive oil in addition to the other foods in your diet? 
 
    -2 
 
       -1 
          0 
        1 
 
     2 
 
  Never 
 
Very Rarely 
 Occasionally 
 Very Often 
 
 Always 
 
 
 
3. On a scale of 1 to 10, how difficult did you find it to eat the tree nuts and olive oil every day?  
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
NOT AT ALL 
MODERATELY 
           VERY 
DIFFICULT  
 
 
   DIFFICULT 
     
 
                  DIFFICULT 
 
 
4. On a scale of 1 to 10, how difficult did you find it to consume the olive oil every day?  
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
NOT AT ALL 
MODERATELY 
           VERY 
DIFFICULT  
 
 
   DIFFICULT 
     
 
                  DIFFICULT 
 
 
 
 
 

 
24 
 
 
 
5. On a scale of 1 to 10, how did eating the tree nuts and olive oil daily make you feel?  
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
NOT GOOD 
NO 
           VERY 
AT ALL 
 
 
 
   DIFFERENCE      
 
                      GOOD 
 
6. On a scale of 1 to 10, how did consuming the olive oil daily make you feel?  
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
NOT GOOD 
NO 
           VERY 
AT ALL 
 
 
 
   DIFFERENCE      
 
                      GOOD 
 
 
7. Did you use the provided recipes to help incorporate the tree nuts and olive oil into your diet?  
 
1 
 
2 
 
3 
 
4 
 
5 
Not at all 
 
Not much 
 
Sometimes 
 
Often  
 
Very often 
 
 
8. On a scale of 1 to 5 (with 5 being the very best I have tasted), how much did you like the flavor 
of the olive oil? 
 
          1 
 
        2 
           3 
          4 
 
            5 
Did not like 
       at all 
 
   Disliked 
  somewhat 
 
Neither liked  
   or disliked 
 
     Liked 
somewhat 
 
       Liked a  
     great deal 
 
 
9. On a scale of 1 to 5 (with 5 being the very best I have eaten), how much did you like the 
walnuts? 
 
          1 
 
        2 
           3 
          4 
 
            5 
Did not like 
       at all 
 
   Disliked 
  somewhat 
 
Neither liked  
   or disliked 
 
     Liked 
somewhat 
 
       Liked a  
     great deal 
 
 
 
 
 

 
25 
 
 
10. On a scale of 1 to 5 (with 5 being the very best I have eaten), how much did you like the 
almonds? 
 
          1 
 
        2 
           3 
          4 
 
            5 
Did not like 
       at all 
 
   Disliked 
  somewhat 
 
Neither liked  
   or disliked 
 
     Liked 
somewhat 
 
       Liked a  
     great deal 
 
 
 
11. On a scale of 1 to 5 (with 5 being the very best I have eaten), how much did you like the 
pistachios? 
 
          1 
 
        2 
           3 
          4 
 
            5 
Did not like 
       at all 
 
   Disliked 
  somewhat 
 
Neither liked  
   or disliked 
 
     Liked 
somewhat 
 
       Liked a  
     great deal 
 
 
12. Will you continue to eat tree nuts and olive oil daily after completing the research study?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
13. Will you continue to eat olive oil daily after completing the research study?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
 
14. If eating tree nuts and olive oil improved your metabolic health, how likely would you be to 
continue to eat tree nuts and olive oil daily?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
 
 
 
 

 
26 
 
 
 
 
 
 
15. If consuming olive oil improved your metabolic health, how likely would you be to continue to 
consume olive oil daily?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
 
 
16. Do you think that you would be able to participate in a study like this one that lasted 3-4 years?  
In other words, would you be able to continue eating the tree nuts and olive oil for 3-4 years?  
 
1
 Yes  
 
2
 No 
 
 
 
If no, why not?  
 
 
 
 
 
 
 
 
 
 
 
 
17. Please describe any specific problems or barriers that you encountered that made it difficult for 
you to eat the tree nuts and olive oil each day.    
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 

 
27 
 
18. Please describe any suggestions you have that would make eating the tree nuts and olive oil 
each day easier for participants in future studies.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
19. Can Dr. Reynolds or someone she designates contact you for future studies?  
 
1
 Yes  
2
 No 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
28 
 
Appendix D. 
 
 
 
 
We would like to ask you some questions about epigenetic age.  Epigenetic age 
is a DNA-based measure that predicts the biological age of your cells. Having an 
accelerated epigenetic age means that the biological age of your cells may be 
more advanced than your chronological age. 
 
 
20. Using the scale below, indicate how much you want to know your epigenetic age: 
 
1 
 
2 
 
3 
 
4 
 
5 
 
Not at all 
 
Not much 
 
Neutral 
 
Somewhat  
 
Very much 
 
 
21. For question #1, why did you select that answer? 
 
 
 
 
 
 
 
 
 
 
 
22. Before beginning this study, if you were told your epigenetic age appeared more advanced 
than your chronological age, do you think you would be more or less motivated to eat the nuts 
and olive oil everyday?  
 
1 
 
2 
 
3 
 
4 
 
5 
 
  Not 
motivated 
at all 
 
Less 
motivated 
 
Neutral 
 
More 
motivated 
 
 Very 
motivated 
 
 
 
 
 
 
 
 

 
29 
 
23. Before beginning this study, if you were told your epigenetic age appeared younger than your 
chronological age, do you think you would be more or less motivated to eat the nuts and olive 
oil everyday?  
 
1 
 
2 
 
3 
 
4 
 
5 
 
  Not 
motivated 
at all 
 
Less 
motivated 
 
Neutral 
 
More 
motivated 
 
 Very 
motivated 
 
 
24. If eating tree nuts and olive oil slowed biological aging, how likely would you be to continue to 
eat tree nuts and olive oil daily?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
25. If consuming olive oil slowed biological aging, how likely would you be to continue to consume 
olive oil daily?  
 
1 
 
2 
 
3 
 
4 
 
5 
  Very 
unlikely 
 
Unlikely 
 
Neutral 
 
Somewhat 
likely 
 
 Very likely 
 
 
26. Do you feel we did a good job explaining the concept of epigenetic aging?  
 
1 
 
2 
 
3 
 
4 
 
5 
Not at all 
 
Not much 
 
Neutral 
 
Somewhat  
 
Very much 
 
 
 
 
 
 
 
 
 
 
 
27. For question #76, can you comment on why you selected that answer?