The Effects of Added Sugar Intake on Brain Blood Flow and Hippocampal Function in Midlife Adults
Phase: NA
Conditions: Aging, Healthy Diet
Sponsor: University of Delaware

Cover Page for Study Protocol 
 
Official Title: The effects of added sugar intake on 
brain blood flow and hippocampal function in midlife 
adults 
ClinicalTrials.gov ID: NCT05211726 
Protocol ID: 1760500 
Date of Document: May 8, 2024  
 
 
 

HUMAN SUBJECTS PROTOCOL 
University of Delaware 
Protocol Title: The effects of added sugar intake on brain blood flow and hippocampal function in 
midlife adults 
 
Principal Investigator 
 
Name: 
Christopher Martens, Ph.D. 
Department/Center: 
Kinesiology& Applied Physiology 
Contact Phone Number: 
(302) 831-7270 
Email Address: 
cmartens@udel.edu 
 
Other Investigators: 
 
Name: 
Shannon Lennon, Ph.D. 
Department/Center: 
Kinesiology & Applied Physiology 
Contact Phone Number: 
302-831-2798 
Email Address: 
slennon@udel.edu 
 
Name: 
Curtis Johnson, Ph.D. 
Department/Center: 
Biomedical Engineering 
Contact Phone Number: 
302-831-4098 
Email Address: 
clj@udel.edu 
 
Name: 
Matt Cohen, Ph.D. 
Department/Center: 
Communications Sciences and Disorders 
Contact Phone Number: 
302-831-7071 
Email Address: 
mlcohen@udel.edu 
 
Investigator Assurance: 
 
By submitting this protocol, I acknowledge that this project will be conducted in strict accordance 
with the procedures described. I will not make any modifications to this protocol without prior 
approval by the IRB. Should any unanticipated problems involving risk to subjects occur during this 
project, including breaches of guaranteed confidentiality or departures from any procedures 
specified in approved study documents, I will report such events to the Chair, Institutional Review 
Board immediately. 
1. Is this project externally funded? x YES □ NO 
 
If so, please list the funding source: (Pending) National Institutes of Health (NIH) Grant P20 
GM113125 
 
2. Research Site(s) 
 
X University of Delaware 
□ Other (please list external study sites) 

3. Project Staff 
Please list all personnel, including students, who will be working with human subjects on this 
protocol (insert additional rows as needed): 
 
Table 3.1 
 
NAME 
ROLE 
HS TRAINING COMPLETE? 
Christopher Martens, Ph.D. 
PI** 
Yes 
Shannon Lennon, Ph.D. 
Co-I 
Yes 
Curtis Johnson, Ph.D. 
Co-I 
Yes 
Matthew Cohen, Ph.D. 
Co-I 
Yes 
Melissa Witman, Ph.D. 
Co-I 
Yes 
Kevin Decker, Ph.D. 
Co-I, Postdoctoral Fellow** 
Yes 
Faria Sanjana, M.S. 
Graduate Student** 
Yes 
Nick Rizzi, M.S. 
Graduate Student** 
Yes 
Theodore DeConne, B.S. 
Graduate Student** 
Yes 
Fiona Horvat, B.S. 
Graduate Student** 
Yes 
Wendy Nichols, R.N. 
Research Nurse 
Yes 
Kristina Davis 
Nutritionist 
Yes 
Zoe Rigas 
Undergraduate 
Yes 
Ryan Pohlig 
Statistician 
Yes 
Nazim Karaca 
REDCap Data Analyst 
Yes 
Ibrahim Malik 
MRI Operator 
Yes 
Catherine Awad 
Research Coordinator** 
Yes 
Leif Boddie 
Undergraduate 
Yes 
** Indicates that this person has been trained to administer the informed consent. 
 
4. Special Populations 
 
This research does not involve any special populations. 
 
5. RESEARCH ABSTRACT Please provide a brief description in LAY language (understandable to 
an 8th grade student) of the aims of this project. 
 
Aging is the primary risk factor for Alzheimer’s disease (AD) which is the most common form of 
dementia and among the fastest growing causes of morbidity and mortality in the United States. The 
risk factors for AD emerge during midlife and are similar to cardiovascular and cerebrovascular 
diseases. The impact of midlife vascular changes on the brain are worsened by poor lifestyle habits, 
including eating a diet that contains a lot of added sugars (e.g., ultra-processed foods containing high 
amounts of fructose). One effect of eating a high sugar diet is an elevation in blood triglycerides (TGs), 
which impairs blood vessel function by causing inflammation; however, it is not known whether eating 
a lot of added sugars affects the blood vessels in the brain. The purpose of this project is to determine 
if there is a link between added sugar intake and brain health in midlife adults. Our hypothesis is that 
eating excess added sugar impairs the structure and function of an area of the brain called the 
hippocampus by increasing plasma TGs and systemic inflammation. To test this, we will have people 
eat a high and low sugar diet for 10 days each (in a random order) and test how each diet affects their 
blood vessel function, the structure of their hippocampus, and their memory performance. We expect 
to show that eating a diet that contains a lot of added sugars worsens brain health compared to a diet 
that contains few added sugars. The data generated from this project will help us better understand 
risk factors for dementia and will be used to support a future grant proposal to the National Institutes 
of Health aimed at lowering added sugar intake in mid-life adults and individuals with mild cognitive 
impairment. 

6. PROCEDURES Describe all procedures involving human subjects for this protocol. Include 
copies of all surveys and research measures. 
 
6.1  Study Design 
 
We will conduct a single-blind, randomized-crossover, controlled feeding study (see Protocol Schema 
on next page). We will provide subjects with 10-days each of a research diet containing low sugar (LS; 
5% of energy) vs. high sugar (HS; 25% of energy) in a random order, separated by a 2-week washout. 
Primary and secondary outcomes will be measured at baseline and at the end of each 10-day 
intervention. 
 
6.2  Schedule of Activities 
Table 6.1 
Screening 
Baseline 
Diet #1 
Diet #2 
Day of Diet 
NA 
NA 
NA 
NA 
0 
7 
9 
10 
10 
0 
7 
9 
10 
10 
Visit Number 
1.1 
1.2 
2.1 
2.2 
3.1 
NA 
NA 
3.2 
3.3 
4.1 
NA 
NA 
4.2 
4.3 
Duration (hours) 
1.5 
1.0 
1.5 
2 
0.5 
--- 
--- 
1.5 
2 
0.5 
--- 
--- 
1.5 
2 
Eligibility Criteria 
Informed Consent 
• 
Demographics 
• 
Medical History 
• 
Menstrual History 
• 
PHQ-9 (depression) 
• 
MRI Safety Form 
• 
• 
• 
• 
COVID-19 Form 
• 
• 
• 
• 
• 
• 
• 
• 
MAQ 
• 
3-day diet record 
 
 
Height 
• 
Body Mass 
• 
measured at home daily 
measured at home daily 
Body Composition 
• 
• 
• 
Vital Signs 
• 
• 
• 
• 
• 
Clinical Labs 
• 
• 
• 
Control Measures 
Family History 
• 
PROMIS Measures 
• 
Sleep Quality 
• 
Food Preferences 
• 
APOE genotyping 
• 
Blood Biomarkers 
• 
• 
• 
Urine collection 
• 
• 
• 
IPAQ 
• 
• 
• 
Sleep Questionnaire 
• 
• 
• 
Sleep Quality 
 
 
 
 
 
 
Appetite Survey 
• 
measured at home daily 
measured at home daily 
24-hr urine collection 
 
 
 
 
Outcom 
24-hr ABPM 
 
 
 
 
 
 
Cognitive Function 
• 
• 
• 
Blood Vessel Tests 
• 
• 
• 
MRI (CVR, MRE) 
• 
• 
• 
Abbreviations: PHQ-9, Patient Health Questionnaire 9; MRI, magnetic resonance imaging; PROMIS, patient-reported outcome 
measurement information system; PSQI, Pittsburgh Sleep Quality Index; APOE, apolipoprotein E; ABPM, ambulatory blood 
pressure monitoring; CVR, cerebrovascular reactivity; MRE, magnetic resonance elastography; MAQ, modifiable activity 
questionnaire. Notation: • indicates measurement will happen on the specified visit;   indicates measurement will happen 
between specified visits. 

6.3  Protocol Schema 
 
This schematic depicts the overall study design and outlines the order of key testing visits and outcome 
measures. Specific details regarding which tests are performed at each visit are described in the 
Schedule of Activities table on the previous page. If a measurement is not performed on a specific 
visit, due to equipment failure and/or timing issues, the measurement may be repeated on another 
visit. More specific details regarding each procedure are included below. The purpose of this study is 
to gain preliminary data for an R01 proposal aimed at improving brain health through dietary 
modification of added sugars. 
 
 
 
6.4. List of Procedures - The order, frequency and visit(s) at which each measurement will be made 
is described in the Schedule of Activities table above. 
 
Eligibility Measures – The following information will be collected to determine eligibility. Some 
measures may be repeated at other time points as noted in Table 6.1. 
 
Informed Consent - Subjects will meet with the PI or designated member of the study team to review 
and sign the informed consent form. Individuals trained to administer the informed consent are listed 
in Table 3.1. The study team member will walk the subject through the consent form and will answer 
any questions that the subject may have prior to providing their written consent. As documentation of 
the informed consent, the subject and the investigator performing the consent will sign (or eSign) the 
informed consent form and a copy of the signed consent form will be given to the subject. 
Subject Demographics – A participant intake form will be used to gather basic information including 
date of birth, sex, race and ethnicity. An equal number of men and women between the age of 50-64 
will be enrolled. Race and ethnicity will be gathered for reporting of basic subject characteristics. 
Medical History – Subjects will provide information about any chronic medical conditions that they 
have been diagnosed with, previous hospitalizations and medical procedures, current prescription and 
over-the-counter medications or supplements, known allergies, health habits (e.g., smoking and 
alcohol use), and any current medical conditions or symptoms. Subjects diagnosed with a chronic 
illness or neurological condition will be excluded. 

Gynecological History – Because of the unique age-range of this demographic, we recognize that a 
small proportion of women may still be pre- or peri-menopausal at the time of screening. Because of 
the small sample size in this initial pilot study and the influence of reproductive hormones on vascular 
function1,2, we will only enroll postmenopausal women in this pilot study to reduce potential variability. 
Female participants will answer questions about their menstrual history to confirm that they are 
postmenopausal. Menopause status (either surgical or nonsurgical) will be confirmed in all women as 
a cessation of menses for >1 year; women taking hormone replacement therapy in the preceding year 
will be excluded from the study3. 
Patient Health Questionnaire 9 (PHQ-9) – A brief, 9-item survey will be used to confirm that subjects 
do not have major depressive disorder, as this can result in cognitive impairment and impact the 
primary study results. Subjects must score < 10 on the PHQ-9 to be eligible for this study. 
MRI Safety Form – Subjects will answer questions about any implanted metal or devices (e.g., a 
pacemaker) as well as feelings of claustrophobia to determine if they are eligible to undergo MRI 
scanning. Because the primary outcomes of this study will be obtained using MRI, subjects must 
qualify for the MRI scan to be enrolled in this study. We will repeat this form before each MRI scan. 
COVID-19 Questionnaire - We will ask subjects about their history with COVID-19, including if they 
were ever hospitalized or experienced severe symptoms, and details regarding vaccination. Subjects 
that previously had a severe case of COVID-19 will be excluded from this study. To avoid any influence 
of an acute inflammatory response on our primary outcomes, we will delay baseline testing on subjects 
who are planning to become vaccinated until 1 week after their final vaccination dose. We will repeat 
this form on each day that subjects visit the lab to confirm that there are no changes. 
Modifiable Activity Questionnaire (MAQ) - Because high levels of physical activity affect vascular 
function and brain health, we will administer the Modifiable Activity Questionnaire (MAQ) at baseline 
and exclude subjects who habitually perform an excessive amount of physical activity. 
3-Day Diet Record - Participants will be asked to record everything they eat, including portion sizes, 
over a 3-day period, including two weekdays and 1 weekend day. This log will be returned during their 
next screening visit. The study nutritionist will analyze the results prior to enrollment. We will only enroll 
individuals with a habitual added sugar intake between 10-15% of total energy intake to ensure that 
our study group is reflective of the average American intake based on the National Health and Nutrition 
Examination Survey (NHANES)4. 
Height and Body Mass – We will measure body height and mass for reporting of basic subject 
characteristics and for calculating body mass index (BMI; kg/m2). Subjects with a BMI < 30 kg/m2 will 
be excluded unless their percent body fat is within normal range (see below). 
Body Composition – Body composition (% body fat) will be determined using bioelectrical impedance 
which uses low-level electrical currents through the body to calculate impedance, which is proportional 
to the amount of body water in adipose tissue. This test will be used primarily as a control variable; 
however, it may also be used to confirm eligibility based on BMI (as indicated by a % body fat > 25% 
for men and >33% for women). A tape measure will be used to capture waist circumference. 
Vital Signs - Blood pressure and resting heart rate will be measured from the non-dominant arm in the 
seated and/or supine position after at least 10 minutes of quiet rest using a semi-automated blood 
pressure device. Repeat measurements will be made (with 2 minutes of quiet rest between recordings) 
until 3 blood pressure values obtained are within 5 mmHg of one another. These values will be 
averaged to determine resting systolic and diastolic blood pressure and pulse pressure. Because we 
are interested in determining the effect of the diets on blood pressure, we will exclude subjects with 
stage 1 or stage 2 hypertension as defined by a systolic BP ≥ 130 mmHg or a diastolic BP ≥ 90 mmHg. 

Screening Blood - Blood will be drawn by the research nurse or trained member of the research team. 
Screening blood will be sent to LabCorp for analysis of standard clinical blood chemistry including: a 
comprehensive metabolic panel, hemoglobin A1C, complete blood count with differential, and a lipid 
panel which will be used for determination of eligibility and for reporting baseline subject 
characteristics. With the exception of hemoglobin A1C which is not expected to change over the short 
duration of this trial, these clinical labs will be repeated at the end of each diet to determine if there 
are any changes in metabolism (see Table 6.2 for complete description of blood markers measured 
at each visit). 
Control Measures – The following information will not be used to determine eligibility but will 
serve as important control information. 
Family History - A family history questionnaire will be used to obtain information regarding family 
history of cardiovascular diseases, cancer, diabetes and Alzheimer’s disease in order to determine if 
family history may account for any differences between diets. 
Patient Reported Outcomes - Subjects will complete the Patient Reported Outcomes Measurement 
Information System (PROMIS) 57-item questionnaire to assess self-reported feelings about physical 
function, anxiety, depression, fatigue, sleep disturbance, social engagement, and pain intensity. 
Sleep Questionnaires - Self-reported sleep quality will be assessed using the Pittsburgh Sleep Quality 
Index (PSQI) and the 17-item Munich Chronotype Questionnaire (MCQ). The PSQI is a 19-item self- 
report tool that assesses healthy sleep metrics over a 1-month time interval. The 17-tiem MCQ 
determines sleep midpoint for work and free days in the last month. 
Sleep Quality - Participants will be provided with an Actiwatch accelerometer watch (Phillips) for an 
objective assessment of sleep quality. Subjects will be asked to wear the device on their non-dominant 
wrist for ten days and return it at their next visit. 
Physical Activity Questionnaire – Subjects will complete a questionnaire using the International 
Physical Activity Questionnaire (IPAQ) to determine physical activity measures. The 27 questions of 
the IPAQ inquiries about physical activity in the last 7 days across 5 domains that relate to occupation, 
transportation, housework, recreational activity, and time spent sitting, 
Food Preference Questionnaire – Subjects will complete a brief questionnaire to determine their food 
preferences. This information will be used by the nutritionist to design specific diets that are catered 
to each subject’s food preferences to improve compliance and to determine their energy needs. 
APOE Genotype – Blood will be sent to LabCorp for determination APOE genotype using a standard 
clinical PCR test (LabCorp; CPT: 814401). 
Blood Biomarkers - Additional blood will be retained by the Neurovascular Aging Laboratory for 
isolation of serum, plasma and peripheral blood mononuclear cells (PBMCs) which will be used for 
assessing markers of oxidative stress, inflammation, mitochondrial function, and additional blood lipid 
factors. 
Urine Collection – Urine will be collected for determination of uric acid, which will be used as a marker 
of adherence to each diet. Urine uric acid is expected to increase during the high sugar diet but not 
during the low sugar diet. 
Home Body Weight and Appetite – Subjects will be provided with an electronic scale and will be asked 
to record their body mass daily, using an electronic (REDCap) survey that will be emailed to them at 
the same time each day to confirm that they are consuming the isocaloric diet and not gaining or losing 
body mass. They will also be asked to rate their perceptions of hunger and satiety using a visual 
analog scale (0-100 mm). 

24-hour Urine Collection – Subjects will collect all of their urine for 24 hours to measure urine sodium 
concentration as a measure of compliance to each diet. 
Outcome Measures – The following measures will be made for testing primary and secondary 
hypotheses. All measures will be made on the same day (e.g., baseline or day 10 of each diet) 
with a short break in between arterial stiffness testing to drive to the MRI center. 
Visits 2.1, 3.2 and 4.2 (Location: Center for Biomedical and Brain Imaging) 
MRI Scan – The MRI scan will include the following tests: 
o 
Cerebrovascular Reactivity (CVR) will be measured as the maximal change (from rest) in total 
cerebral perfusion during 3-minutes of hypercapnia (+9 mmHg increase in PETCO2) using pseudo- 
continuous arterial spin labeling (PC-ASL)6, which uses radio-frequency pulses to magnetically 
label the water in the blood as an endogenous tracer 6 and thus can be performed without the 
need for a contrast agent. Hypercapnia will be induced using an MR-compatible prospective end- 
tidal targeting system which allows for precise and repeatable manipulation of PETCO2 on a breath- 
by-breath basis through a face mask using a computerized gas blender (RespirActTM)7-9. Beat-to- 
beat BP will be assessed using an MRI-compatible non-invasive BP (NIBP) monitor (Biopac, 
Goleta, CA) to normalize blood flow to mean arterial blood pressure. 
 
o 
Brain Microstructural Integrity will be assessed by Magnetic Resonance Elastography (MRE) of 
the brain. During the acquisition of MRE images, the subject’s head will be vibrated using the 
Resoundant Acoustic Driver System. The Resoundant is an FDA-approved device for inducing 
small vibrations in a scan subject. The device consists of a small, pillow-like pad that is attached 
in the MRI head coil and that the subject’s head rests on. This pad receives vibrations from tubing 
that attaches to an active driver placed in the control room. The vibrations received result in a 
small up-down vibration of the subject’s head. The active driver is activated in concert with the MR 
scanner and is controlled by the investigator performing the scan. 
 
Structural T1- and T2-weighted and BOLD MRI images will be acquired following MRE and ASL 
acquisitions, which are necessary for comparing MRE and ASL findings with brain structure and 
activity. There will be no vibration during these additional scans. During all scans the subjects will wear 
disposable earplugs and/or headphones to reduce scanner noise. Participants may also be asked to 
wear a pulse oximeter on their finger to measure heart rate or a respiratory belt around their chest to 
measure breathing. This is to observe any potential MRE signal changes from movement associated 
with breathing or heart rate. Both devices are provided by Siemens with the MRI scanner, are MR- 
safe and MR-compatible, and interface with the MRI scanner software. 
Visits 2.2, 3.3 and 4.3 (Location: Neurovascular Aging Laboratory) 
24-Hour Ambulatory Blood Pressure (ABPM) – Subjects will be asked to wear a blood pressure 
monitor on their upper arm for 24 hours at baseline and during the final day of each diet. The blood 
pressure cuff will be set to automatically take their blood pressure every 20 minutes during the day 
and every 30 minutes at night. If possible, subjects will be instructed to sit down and relax when they 
feel the upper arm blood pressure start to inflate. They will also be asked to refrain from caffeine, 
alcohol, and exercise on this day. 
Arterial Stiffness – The following measures will be made to characterize arterial stiffness. 
o 
Pulse Wave Velocity (PWV) – We will use transcutaneous tonometry to measure the pulse in the 
carotid artery while placing a standard blood pressure cuff around the thigh to measure the pulse 
in the femoral artery. The pressure wave at each recording site will be aligned to the cardiac cycle 

using a 3-lead ECG and PWV will be calculated as the distance between measurement sites 
divided by the transit time of the arterial pulse wave. 
o 
Pulse Wave Analysis – We will derive central (aortic) pulse pressure (PP), augmentation pressure 
(AP) and augmentation index (AI) from the radial artery pulse wave using applanation tonometry. 
o 
Carotid artery compliance – We will record a high-resolution ultrasound video of the carotid artery 
and use applanation tonometry to determine carotid artery pulse pressure. Compliance will be 
calculated as the change in carotid diameter for a given change in pressure. 
Brain Blood Vessel Test - A small ultrasound probe will be placed on the top of the skin above the 
temple, and we will record how fast the blood is moving through a major artery in the brain. At the 
same time, we will measure the amount of carbon dioxide (CO2) in the air that is exhaled by having a 
soft flexible tube in the front of the nose with ~1 cm extensions that are inserted into the tips of the 
nostrils and respiration will be measured using a flexible respiration strap. This brain blood vessel test 
will be measured in response to an acute (~30 second) breath hold, as previously described5. The 
participant will be monitored for signs of distress and the test will be stopped immediately if they 
express discomfort. The breath-hold test is self-guided and the participant is free to breathe at any 
time. 
Cognitive Function – Memory function will be assessed by performance on the Hopkins Verbal 
Learning Test (HVLT-R), The Brief Visuospatial Memory Test (BVMT-R), and the NIH Toolbox Pattern 
Comparison and Flanker tests. The HVLT-T and BVMT-R have multiple versions to prevent learning 
tests. We will choose three versions of this test at the beginning of the trial and will administer a 
different version at each visit. The order will be counterbalanced to avoid any effect of test version on 
cognitive outcomes. The selected NIH Toolbox texts are less prone to learning effects. 
 
Study Restrictions 
Subjects must avoid over-the-counter medications for 48 hours, vigorous aerobic exercise or alcohol 
for 24 hours, and all food, supplements and caffeine for ≥ 12 hours before testing visit but will be 
allowed to consume water. There are no restrictions for Visit 1.1, unless this visit is combined with 
Visit 1.2, in which case the above restrictions apply. 
 
Table 6.2. Table of Blood Tests 
 
Test # 
Type 
Volume 
Visit 1.2 
Visit 3.3 
Visit 4.3 
Screening Labs 
Comprehensive 
Metabolic Panel 
322000 
SST 
Serum 
4 mL 
X 
X 
X 
Complete Blood 
Count 
005009 
EDTA 
Plasma 
4 mL 
X 
X 
X 
Lipid Panel 
303756 
SST 
Serum 
4 mL 
X 
X 
X 
Control Measures 
Hemoglobin A1C 
001453 
EDTA 
Plasma 
4 mL 
X 
--- 
--- 
APOE genotype 
504040 
EDTA 
Plasma 
4 mL 
X 
--- 
--- 
Blood Biomarkers 
Oxidative 
Stress/Inflammation 
--- 
EDTA 
Plasma 
4 mL 
X 
X 
X 

--- 
SST 
Serum 
4 mL 
X 
X 
X 
PBMC’s 
--- 
EDTA 
Plasma 
32 mL 
X 
X 
X 
Total Per Visit 
60 mL 
(4TBL) 
52 mL 
(3.5 TBL) 
52 mL 
(3.5 TBL) 
Total Volume for Study (mL) 
164 mL 
(11 TBL) 
Total Volume for Study (pints) 
1/3 pint 
 
 
 
 
6.5. Description of Research Diets 
 
The study nutritionist will prepare two 10-day isocaloric research diets for each subject. The diets will 
differ in the amount of energy from added sugar sources. Added sugars include food products 
containing syrups and other caloric sweeteners that are added during processing or preparation of 
food. The majority of added sugars in the United States come from sucrose (table sugar) and products 
containing high-fructose corn syrup; however other sugars are also used during production or 
preparation of foods which are considered added sugars. Sugars found naturally in fruits, vegetables 
and dairy products that have not been added during processing or preparation are not considered 
added sugars. An example menu of each diet and the corresponding nutrient breakdown is presented 
in Table 6.3. The specific amount of added sugar in each research diet was selected to maximize the 
ability to detect changes in vascular function and brain health while also delivering a diet that is 
representative of current American diets. Data from the National Health and Nutrition Examination 
Survey (NHANES) suggest that only half of all Americans meet the current the guidelines for added 
sugar intake4, which have been set by regulatory bodies including the American Heart Association 
(AHA), US Department of Agriculture (USDA) and the World Health Organization (WHO) to consume 
less than 10% of all calories from added sugars10,11. Although the average intake of added sugars (as 
a percentage of total calorie intake) in the United States is ~13%, those who meet current guidelines 
consume an average of ~5% of total energy from added sugar, whereas the average intake for those 
not meeting the guidelines is >20%4. Accordingly, the chosen amount of added sugar in the low (5% 
of energy) and high (25% of energy) added sugar diets are within the range of what many Americans 
are eating and have a high likelihood of modulating blood lipid profiles, thus enabling us to examine 
the effect of dietary added sugars on important physiological functions indicative of brain health. 
 
 
Table 6.3. Example Menus 
5% Added Sugar 
25% Added Sugar 
Breakfast 
Instant oatmeal with 2% milk and raisins 
Coffee with sugar and cream 
Snack 
Popcorn and peanut butter crackers 
Lunch 
Turkey sandwich with wheat bread, American 
cheese and mayonnaise 
Mini pretzels and apple 
Snack 
Tortilla chips, Baby carrots and Hummus 
Breakfast 
Pancakes with syrup 
Cranberry juice cocktail 
Snack 
Coffee Cake 
Lunch 
Chicken noodle soup, saltine crackers 
String cheese 
Snack 
Coke-a-cola and Dry roasted almonds, 

Dinner 
Boneless skinless chicken breast, baked potato and 
green beans 
Oreo cookies 
Dinner 
Spaghetti and tomato sauce with ground turkey 
and parmesan cheese 
Nutrient Breakdown 
Carbohydrates (%) 
55 
Carbohydrates (%) 
55 
Fat (%) 
30 
Fat (%) 
30 
Protein (%) 
15 
Protein (%) 
15 
Total Calories 
1,944 
Total Calories 
1,991 
Total Carbohydrates (g/day) 
271 
Total Carbohydrates (g/day) 
285 
Total Sugar (g/day) 
77 
Total Sugar (g/day) 
157 
Added Sugar (g/day) 
25 
Added Sugar (g/day) 
131 
Added Sugar (% Kcal) 
5 
Added Sugar (% Kcal) 
25 
Fiber (g/day) 
26 
Fiber (g/day) 
12 
 
Diet Adherence 
 
During the strictly controlled diet periods, all meals and snacks will be provided to the subjects. Food 
for the 10-days will be packed in a cooler and picked up by the subjects at Visits 3.1 and 4.1. All food 
preparation will be overseen by our registered dietitian and prepared in the metabolic kitchen in the 
Tower at STAR. Energy needs will be calculated at baseline using the Mifflin-St. Jeor equation11 and 
will be corrected for habitual physical activity in order to ensure weight stability throughout the study. 
Subjects will be asked to consume only the provided food. No other food should be eaten during either 
of the 10-day diet periods. Subjects will be asked to report extra food items using the “Extra Food Log” 
form as well as bring back any uneaten food to be weighed. If it is determined that a subject is eating 
“outside foods” (>50 kcal on 2 or more days), they will be terminated from the study. The total calories 
provided are designed to keep the subjects weight stable during this period (this is referred to as 
“isocaloric”). We will also provide them with water (bottled) to drink during each diet. Subjects will 
report to the lab on the 10th day of each diet to undergo all post-intervention testing (Visits 3.2, 3.3, 
4.2, & 4.3). 
Washout Period 
 
Subjects will complete a 2-week washout period in between each diet to ensure enough time for lipid 
markers and physiological functions to return to baseline values between diets. Previous dietary 
intervention crossover studies over a 3-6 week period without a washout period have not reported 
carryover effects on measures of large elastic artery stiffness, vascular function or cognitive 
performance13,14. Accordingly, we expect the proposed measures of physiological function to return to 
baseline quickly and the proposed 2-week washout period represents a conservative approach. 
 
 
7. STUDY POPULATION AND RECRUITMENT 
 
Study Population – The target enrollment for this study is 200 healthy adult men and women between 
50-64 years of age, with a target completion of 40 participants. This age group has been defined by 
NIH as being at high risk of accumulating cardiometabolic risk factors that preclude chronic age-related 
diseases15-17. To avoid any influence of baseline diet (e.g., chronic consumption of a high-sugar diet 
prior to enrollment), will only enroll those with a habitual intake of added sugar that is reflective of the 
average American intake (~13% of total calories) based on the National Health and Nutrition 
Examination Survey (NHANES)18. This will increase the likelihood of observing changes in our primary 
outcome variables in response to the proposed research diets which are designed to deviate from 
baseline proportion of added sugars by about 10% of calories in each direction. Importantly, even if 

subjects are close to the upper or lower cutoff, we are still likely to observe changes in primary 
outcomes when placed on the opposite arm of the crossover. Because this is a pilot study, we will 
analyze the data after the first 5-10 participants have completed the trial and ensure that we are seeing 
an effect of the diets on the primary outcome variables. If we do not see an effect, we will amend the 
protocol to increase the duration and/or modify the composition of the diets. 
To be eligible to participate, volunteers must meet the following specific enrollment criteria: 
Inclusion Criteria: 
• 
ability to provide informed consent; 
• 
men and postmenopausal women aged 50-64 years; 
• 
habitual intake of added sugars ≤15% of total calories18; 
• 
systolic BP < 130 mmHg; diastolic BP < 90 mmHg19; 
• 
body mass index (BMI) <30 kg/m2 and % body fat < 25% for men and < 33% for women 
• 
fasting triglycerides < 200 mg/dl (< 2.3 mmol/L)20; 
• 
LDL cholesterol <160 mg/dl (4.14 mmol/L) 21 
• 
fasting plasma glucose <126 mg/dl (<7.0 mmol/L) and hemoglobin A1C < 6.5% at screening22; 
• 
weight stable in the prior 6 months (≤ 2 kg weight change); 
• 
blood chemistries indicative of normal liver enzymes and renal function (estimated glomerular 
filtration rate using the MDRD prediction equation must be >60 ml/min/1.73 m2) 
Exclusion Criteria: 
• 
current use of medications or supplements known to lower blood triglycerides or cholesterol 
(e.g., fibrates, statins, high dose niacin, high dose omega-3 supplement); 
• 
chronic clinical diseases (e.g., coronary artery/peripheral artery/cerebrovascular diseases, heart 
failure, diabetes, chronic kidney disease requiring dialysis, neurological or autoimmune 
conditions affecting cognition (e.g. Alzheimer’s disease or other form of dementia, Parkinson’s 
disease, epilepsy, multiple sclerosis, large vessel infarct); 
• 
major psychiatric disorder (e.g. schizophrenia, bipolar disorder); 
• 
major depressive disorder (PHQ-9 ≥ 10); 
• 
current or past (i.e., last 3 months) use of anti-hypertensive or other cardiovascular-acting 
medications known to influence vascular function and/or arterial stiffness23.24; 
• 
current medication use likely to affect CNS functions (e.g. long active benzodiazepines); 
• 
concussion within last 2 years and ≥ 3 lifetime concussions25; 
• 
heavy alcohol consumption (defined by the CDC and USDA as ≥8 drinks/week for women and 
≥15 drinks/week for men)26. 
• 
claustrophobia, metal implants, pacemaker or other factors affecting feasibility and/or safety of 
MRI scanning; 
• 
recent major change in health status within previous 6 months (i.e., surgery, significant infection 
or illness); 
• 
current smoking within the past 3 months. 
• 
High degree of physical activity as defined by ≥ 25 leisure MET-hours/week, within the past 3 
months. 
 
Recruitment 
Subjects will be recruited from Newark, DE and the surrounding area. Recruitment methods may 
include posted flyers, digital advertisements (e.g., Facebook), direct mailings to age-specific regional 
mailing lists, UD alumni and employee mailing lists, UD press releases, information sharing with a 

variety of minority organizations associated with UD and the Newark community, participation in health 
fairs, and emails sent to past participants interested in being recontacted about new research studies. 
Primary Exit Criteria 
 
• 
Completion of the study; 
• 
Failure to eat the diet during the dietary trial; 
• 
Eating foods other than those prepared for the subject during the dietary intervention (>50 
kcal/day on 2 or more days); 
• 
Not showing up to the agreed upon study visit two times or chronically canceling within 2 hours 
of the agreed upon visit (two times); 
• 
Failure to complete MRI scanning (e.g., due to claustrophobia); 
• 
Donating blood or a large volume of blood drawn within 6 weeks of study participation or 
during the study (subjects should not donate blood in 6 weeks following study participation). 
 
Subjects will also have the option of withdrawing from the study at any time for any reason. The 
number of subjects exiting the study and the reasons for exit will be carefully documented. Subjects 
who withdraw from the study will be replaced in order to achieve the sample size required for 
appropriate statistical power. 
 
8. RISKS AND BENEFITS 
List all potential physical, psychological, social, financial or legal risks to subjects (risks listed here 
should be included on the consent form). 
 
High Sugar Diet – There is minimal risk associated with consuming a high sugar diet for this duration. 
Each diet will be specifically catered to each subject, taking into account food preferences and any 
known food allergies. The diets are designed to be isocaloric and are not expected to change body 
weight. In the event of reported weight loss or reports of hunger, diets will be adjusted to increase 
energy intake. Unlike high amounts of glucose, 90% of dietary fructose intake is metabolized by the 
liver and does not substantially increase blood glucose levels27; however, we do expect the high-sugar 
diet to raise plasma TGs and there is a possibility that it will also raise other cardiometabolic risk factors 
(e.g., LDL-cholesterol)28-29. We expect these parameters to return to baseline after participants return 
to their habitual diet. Moreover, there is no evidence that the short-term dietary changes proposed in 
this study influence long-term risk of cardiometabolic disease or cognitive impairment. Importantly, our 
high-sugar diet is representative of what a large number of adults in the United States are already 
consuming on a daily basis4,18. 
Venous Catheter/blood draw – Discomfort associated with insertion of the needle; local bleeding and 
a small hematoma (~10% of cases); risk of blood clot (~1% of cases); risk of infection of a hematoma 
or significant external blood loss (<1 in 1000); risk of fainting. 
Genetic Risk for AD – One of the blood tests will determine whether or not the subject carries a gene that 
has been associated with risk for Alzheimer’s disease (AD). People with this gene have a higher chance 
of developing AD compared with people who do not carry the gene. Some people who carry the gene 
may never develop AD. Others may develop AD even though they do not carry the gene. Subjects will 
be provided with the results of this test upon request only. 
24-hour ABPM – There are no known risks associated with wearing the 24-hour blood pressure 
monitor. Subjects may find it irritating as it inflates regularly. 
Acute Hypercapnia (CO2 inhalation) – There are no risks associated with inhalation of small amounts 
of carbon dioxide. The hypercapnia protocol has been used in human studies clinically and in the 
research setting for decades without any adverse events. Study subjects may experience an increase 

in heart rate, flushed skin, minor disorientation, or develop a mild headache during hypercapnia. Some 
subjects may notice a slight metallic taste when breathing the CO2 gas mixture. 
MRI Scan – MRI is an imaging technique that uses radio waves and magnetic fields to produce images 
of internal structures in the body. Unlike X-rays, the MRI does not use any ionizing radiation, and it 
does not use radioactivity, so there is no radiation related risks from having an MRI scan done. Below 
there is a description of MRI related risks and what is being done to reduce any possible risks 
associated with them: 
• 
Metal – The MRI scanner produces a constant strong magnetic field, which may cause any metal 
implants, clips, or implanted medical devices within the body to shift position or malfunction. 
Subjects will be screened prior to entering the MRI scanner and those with implanted metal, clips 
or devices that are not MR-compatible will be excluded from this study. Metallic objects brought 
into the MRI environment can become hazardous projectiles and can also interfere with the data 
quality. To minimize this risk, subjects will be asked to remove all metal jewelry, belts with metal 
buckles, and all items from the pockets, including coins, electronics (including cell phones and 
hearing aids) and wallets. Some subjects may be asked to change into a gown if their clothing 
contains significant metal, including metal underwire bras. 
• 
Inner ear damage – MRI scanning produces loud noises that can cause damage to the inner ear 
if appropriate hearing protection is not used. Subjects will wear earplugs and headphones to 
protect their ears. 
• 
Claustrophobia – The subject’s head will be centered inside a close-fitting scanning coil positioned 
in the bore of the scanner. If the subject feels anxious in confined, spaces they may not want to 
participate. If they are unsure, they can try the “mock” scanner at the MRI facility to evaluate their 
comfort level with the enclosed space of the magnet bore. If they decide to participate and begin 
to feel claustrophobic, they will be able to communicate with the research team via the intercom 
or the squeeze ball and the test will be discontinued. 
• 
Burns – In rare cases, contact with the MRI transmitting and receiving coil, conductive materials 
such as wires or other metallic objects, or skin-to-skin contact that forms conductive loops may 
result in excessive heating and burns during the experiment. The operators of the MRI scanner 
will take steps, such as using foam pads when necessary, to minimize this risk. Tattoos with 
metallic inks can also potentially cause burns. Subjects will be instructed to let the MRI operator 
know immediately if they experience any heating or burning sensations during a scan. The 
scanning session will be stopped as soon as they tell the operator. 
• 
Nerve or muscle stimulation – While the scanner is operating, there is a small chance that the 
rapidly changing magnetic fields could cause a slight tingling sensation or a muscle twitch, usually 
felt in the upper arms or torso. While these sensations may be startling, they are not dangerous or 
a health risk, and they have no lasting consequences. The sensations should stop when the scan 
ends. Because these sensations may nevertheless be distracting or even possibly uncomfortable, 
subjects will be instructed to squeeze the signal bulb to alert the scanner operator if they feel 
uncomfortable tingling or muscle twitching and we will immediately stop the scan. They will then 
have the opportunity to withdraw from the study or to continue. 
• 
Magnetic Resonance Elastography: This study uses elastography to study the mechanical 
properties of the brain. Elastography uses low-amplitude (tens of microns) vibration at 50 Hz 
applied to the head to generate shear waves in the brain. Vibration is applied for less than 10 
minutes of total duration. There is no known risk to vibration exposure at this amplitude, frequency, 
and time. However, it is possible the vibration may cause the subject to feel slight discomfort, and 
they will be instructed to alert the technologist via intercom or by squeezing the emergency ball if 
this occurs. The scan and vibration will be stopped immediately. 

In your opinion, are risks listed above minimal* or more than minimal? If more than minimal, 
please justify why risks are reasonable in relation to anticipated direct or future benefits. 
(*Minimal risk means the probability and magnitude of harm or discomfort anticipated in the research are not greater 
than those ordinarily encountered in daily life or during the performance of routine physical or psychological 
examinations or tests) 
 
The risks associated with all procedures in this protocol are minimal and the magnitude of harm or 
discomfort are not greater than those encountered during similar procedures completed as part of 
routine clinical care (e.g., phlebotomy, MRI scanning). Although patients receiving a clinical MRI 
scan do not typically undergo the MRE and hypercapnia protocols, the overall risks and discomforts 
associated with these procedures are extremely low. 
 
What steps will be taken to minimize risks? 
 
The potential risks of the proposed study will be minimized by: 
• 
using only safe, well-established procedures with only qualified and experienced personnel 
performing the procedures; 
• 
having a registered dietician prepare and package all food in a dedicated research kitchen; 
• 
monitoring any gastrointestinal discomfort associated with consuming the diet; 
• 
ensuring constant personal monitoring of each experimental session by the investigators; 
• 
screening all participants for ability to undergo an MRI; 
 
Describe any potential direct benefits to participants. 
 
Because the risks of participating in this study are relatively small, the risk-to-benefit ratio also is 
relatively small. Subjects will receive benefits associated with overall knowledge of their health from 
the extensive testing performed for screening purposes and the established subject characteristics 
(i.e., blood glucose status, plasma lipids and lipoprotein profile, blood pressure, blood chemistries). 
 
Describe any potential future benefits to this class of participants, others, or society. 
 
The findings from the proposed research will provide important new information regarding the 
influence of dietary intake of added sugar on cardiovascular and brain health in midlife adults. In 
addition, the proposed research will also provide important insight into the potential mechanisms 
(e.g., TGs, inflammation) by which added sugar may negatively impact brain health. This information 
will contribute to the knowledge of how to prevent and treat cerebrovascular and cognitive 
impairment in midlife adults and may lead to future insight into preventing Alzheimer’s disease risk. 
 
If there is a Data Monitoring Committee (DMC) in place for this project, please describe when 
and how often it meets. 
 
There is no DMC for this study. 
 
 
9. COMPENSATION 
 
Will participants be compensated for participation? Yes 
If so, please include details. 

Subjects will receive $200 in total compensation for completing this study. They will receive $100 for 
completing the first diet and $100 for completing the second diet (including the laboratory visits). If 
they drop out after the screening visit (Visit 1.2), they will not be compensated because subjects will 
be receiving medical information from all of the screening tests free of charge. If they consume the 
prepared food and complete the 24-hour blood pressure monitoring but do not complete the 
laboratory visit, they will receive half the amount for that diet trial ($50); however, if this occurs during 
the first diet, the investigators reserve the right to terminate the subject depending on the reason for 
not completing the laboratory visit. Compensation does not include travel time, but upon request will 
compensate subjects for their travel mileage, at the current IRS approved mileage rate. 
Compensation is based on completion of study visits, not individual procedures. Subject can be 
compensated an additional $20 for each subject referral that is consented into this study. Table 10.1 
outlines the total compensation for each visit is presented below. 
Table 10.1 Compensation Schedule 
 
Visit # 
Duration  
Purpose 
Total 
Phase 1 
Visit 1.1 & 1.2 
2.5 hr 
Informed Consent & 
Screening Measures 
No compensation 
Subjects receive free 
health information 
Phase 2 
Visit 2.1 
2 hr 
Vascular and Cognitive 
Function 
$50 (only if visit 3.2 & 3.3 
is not completed) 
Visit 2.2 
1.5 hr 
MRI 
Phase 3 
Visit 3.1 
30 min 
Diet Pick-Up 
$100 
Visit 3.2 
2 hr 
Vascular and Cognitive 
Function 
Visit 3.3 
1.5 hr 
MRI 
Phase 4 
Visit 4.1 
30 min 
Diet Pick-Up & Blood Draw 
$100 
Visit 4.2 
2 hr 
Vascular and Cognitive 
Function 
Visit 4.3 
1.5 hr 
MRI 
Total Duration 
14 hr 
Total Compensation 
$200 
 
 
 
10. DATA 
 
Will subjects be anonymous to the researcher? No. 
 
 
If subjects are identifiable, will their identities be kept confidential? (If yes, please specify 
how) 
 
Subjects will not be individually identified, except by a subject ID number known only to the 
investigators. 
 
How will data be stored and kept secure (specify data storage plans for both paper and 
electronic files. For guidance see http://www.udel.edu/research/preparing/datastorage.html ) 
Physical data will be stored in a locked file cabinet in the PI’s lab space and will only be accessible by 

members of the research team. Physical data that are transcribed into an electronic database, and 
electronically captured data will be kept on a password protected laboratory server that is maintained 
by the University of Delaware. Electronic data will be periodically backed-up onto an external hard 
drive that will be kept in a locked office space. All of the ultrasound images and files generated by the 
SphygmoCor and RespirAct devices will be coded with an identification number and will not contain 
the name of the subject. The names of subjects will not be identified in any publication arising from 
these studies. Only the PI and research staff will have access to data from this study. 
 
How long will data be stored? All data will be stored in a locked cabinet or a password protected 
computer indefinitely. 
 
Will data be destroyed? □ YES X NO (if yes, please specify how the data will be destroyed) 
 
Will the data be shared with anyone outside of the research team? X YES □ NO (if yes, please 
list the person(s), organization(s) and/or institution(s) and specify plans for secure data transfer) 
 
The data may be shared with the National Institutes of Health. The trial will be registered with 
clinicaltrials.gov once the funding is finalized; however, only de-identified data will be reported. 
 
How will data be analyzed and reported? 
 
Data will be aggregated and reported as group means or as de-identified, individual data points. 
Because this study is intended to support a larger R01 proposal, we will analyze the data after the first 
5-10 subjects have completed all testing. If we do not observe any effect of the diet on primary 
outcomes, we will consider amending the protocol to increase the duration of the controlled feeding 
window and/or modify the composition of the diets. Any subjects tested prior to a major amendment 
of the protocol will be replaced to achieve the target sample size. 
 
 
11. CONFIDENTIALITY 
Will participants be audiotaped, photographed or videotaped during this study? 
 
Subjects will be audiotaped for the purpose of analyzing their memory tests after each visit. These 
recordings will be deleted immediately after the test has been scored. 
 
How will subject identity be protected? 
 
Information obtained from this study will be kept strictly confidential. Subjects will not be individually 
identified, except by subject number, known only to the investigators. All data stored as paper files or 
digitally will be kept indefinitely. The paper files are stored in a locked cabinet. While the results of the 
research may be published, subjects’ names and identities will not be revealed. 
 
Is there a Certificate of Confidentiality in place for this project? (If so, please provide a copy). 
 
This study is (pending) funding from NIH which automatically issues a CoC per the notice of award. 
 
 
12. CONFLICT OF INTEREST 
(For information on disclosure reporting see: http://www.udel.edu/research/preparing/conflict.html ) 
 
Do you have a current conflict of interest disclosure form on file through UD Web forms? 

Yes. 
 
Does this project involve a potential conflict of interest*? 
 
* As defined in the University of Delaware’s Policies and Procedures ,a potential conflict of interest (COI) occurs 
when there is a divergence between an individual’s private interests and his or her professional obligations, such that 
an independent observer might reasonably question whether the individual’s professional judgment, commitment, 
actions, or decisions could be influenced by considerations of personal gain, financial or otherwise. 
 
 
If yes, please describe the nature of the interest: 
 
 
13. CONSENT and ASSENT 
 
  X   Consent forms will be used and are attached for review (see Consent Template under Forms 
and Templates in IRBNet) 
 
 
 
 Additionally, child assent forms will be used and are attached. 
 
 
 
 Waiver of Documentation of Consent (attach a consent script/information sheet with the 
signature block removed). 
 
 
 
 Waiver of Consent (Justify request for waiver) 
 
 
14. Other IRB Approval 
Has this protocol been submitted to any other IRBs? 
 
 
If so, please list along with protocol title, number, and expiration date. 
 
 
15. Supporting Documentation 
Please list all additional documents uploaded to IRBNet in support of this application. 
 
Informed Consent Form 
 
Screening and Control Measures – the uploaded forms are examples only and the format may be 
modified for online administration via REDCap. 
 
- 
COVID19 Form 
- 
Daily Appetite Survey 
- 
Diet Recall Form 
- 
Family History 
- 
Food Preference Form 
- 
Intake and Demographics Form 
- 
International Physical Activity Questionnaire (IPAQ) 
- 
Medical History Form 

- 
Menstrual History Form 
- 
Modifiable Activity Questionnaire (MAQ) 
- 
MRI Screening Form 
- 
Munich Chronotype Questionnaire 
- 
Pittsburgh Sleep Quality Index (PSQI) 
- 
PHQ-9 Depression Survey 
- 
PROMIS-57 Profile v2.1 
 
Outcome Measures – the uploaded forms are examples only and the format may be modified for 
online administration via REDCap or using an iPad (e.g., NIH Toolbox) 
 
- 
Brief Visuospatial Memory Test (BVMT-R) 
- 
Hopkins Verbal Learning Test (HVLT-R) 
- 
NIH Toolbox 
o 
Pattern Comparison Processing Speed Test 
o 
Flanker Inhibitory Control Test 
Advertisements 
 
- 
novaLab_Sugar Feeding Study_ Email and Mailed Letter 
- 
novaLab_Sugar Feeding Study_Recruitment Flyer 
- 
novaLab_Sugar Feeding Study_Udaily Bulletin & Social Media Ad 

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