Intermittent Calorie Restriction, Insulin Resistance, and Biomarkers of Brain Function
Phase: NA
Conditions: Alzheimer's Disease, Obesity, Diabetes Mellitus
Sponsor: National Institute on Aging (NIA)

PI name: Dimitrios Kapogiannis 
 
Date: 07/27/2016 
Protocol number: 15-AG-0123 
Version: 3 
CNS IRB Protocol Template (rev 1.27.14) 
 
 
  
Protocol Title: Intermittent Calorie Restriction, Brain Insulin Resistance, and Biomarkers 
of Brain Function  
Abbreviated Title: Calorie Restriction and Brain Insulin Resistance 
Protocol Number: 15-AG-0123 
NCT Number: NCT02460783 
Date of This Submission/Version: 07/27/2016 
 
Principal Investigator 
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
 
Dimitrios Kapogiannis, M.D. LNS/NIA. Harbor Hospital. 410-350-3953. 
kapogiannisd@mail.nih.gov 
 
Lead Associate Investigator  
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
Seema Gulyani, Ph.D., CRNP, LNS/NIA. Harbor Hospital. 410-350-7370.  
 
Associate Investigators (list all) 
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
Mark P. Mattson, Ph.D. LNS/NIA. BRC. 410-558-8463. mark.mattson@nih.gov 
Josephine M Egan, M.D., LCI/NIA. Harbor Hospital. 410-350-3955. eganj@grc.nia.nih.gov 
Tamara Harris, M.D., M.S. LEDB/NIA. BRC. 301-496-6044. harrista@nia.nih.gov 
Toshiko Tanaka, Ph.D, TGB/NIA. Harbor Hospital. 410-558-8650. toshikota@mail.nih.gov 
Luigi Ferrucci, M.D., Ph.D., TGB/NIA. BRC. 410-558-8110. FerruciLu@grc.nia.nih.gov 
Roger Mullins, MA, Ph.D, LNS/NIA.  Harbor Hospital. 410-350-3953. 
 
Research Contact  
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
 
Erondu Onyinye, R.N. LCI/NIA. Harbor Hospital. 410-350-7319. onyinye.erondu@nih.gov  
 
Medical Advisory Investigator  
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
 
Dimitrios Kapogiannis, M.D. LNS/NIA. Harbor Hospital. 410-350-3953. 
kapogiannisd@mail.nih.gov  
 
Total requested accrual (separately specify planned accrual for each subject group)     
 
  (0) Patients       
  (40) Healthy Volunteers (We request permission to screen up to 100 potential 
participants, in order to allow for 60 potential screen failures/dropouts; and 40 
completers) 
 
  
 

 
 
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Project Uses Ionizing Radiation: No  x Yes (attach RSC/RDRC documentation) 
  
 
   Medically-indicated only 
  
 
x  Research-related only 
  
 
   Both 
 
IND/IDE  
 
 
x No   
oYes (attach FDA documentation) 
  
Drug/Device/#_____________________ 
  
Sponsor: __________________________ 
 
Durable Power of Attorney  
 
x No   
o Yes  
 
Multi-institutional Project 
 
 
x No   
o Yes  
 
 
 
 
 
 
 
Institution#1_NIA/NIH________________    FWA #_________  
Date of IRB approval ______________(attach IRB documentation) 
 
 
 
Institution#2_________________    FWA #_________  
 
Date of IRB approval ______________(attach IRB documentation) 
 
Data and Safety Monitoring Board  
x No   
o Yes  
 
Technology Transfer Agreement  
 
x No   
o Yes  
Agreement type and number __________________ Expiration Date_____________ 
 
Samples are being stored  
 
 
o No 
  
x Yes  
 
Flesch-Kincaid reading level of consent form: 9.7  

 
 
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PRECIS 
 
Being overweight or obese can cause insulin resistance (IR), which is defined as reduced 
responsiveness to insulin by the cells of various tissues or organs. IR at midlife increases 
the risk of developing Alzheimer’s disease (AD). We recently discovered novel 
biomarkers of brain IR (altered Tyr and Ser phosphorylated forms of insulin receptor 
substrate 1; IRS-1) in plasma exosomes enriched for neuronal origin.  Moreover, IR is 
associated with AD biomarkers including deficits in resting state brain activity and 
cognitive performance. Calorie restriction is defined as consuming fewer calories than 
what is considered normal without a lack of nutrients. A certain type of calorie 
restriction, in which one consumes 500-600 calories a day for two consecutive days, 
followed by non-restricted eating for 5 days (5-2 CR), has been shown to lower 
peripheral insulin resistance effectively. Effects of CR and lowering peripheral IR on 
brain IR and cognition are unknown. The goal of this exploratory pilot study is to provide 
proof-of-concept that 5-2 CR at midlife can reverse brain IR, lower peripheral IR, 
improve cognitive performance, and increase brain activation at rest and during tasks. 
Specifically, we will study the effects of 8 weeks of 5-2 CR versus a control dietary 
intervention on brain and peripheral IR, memory and executive function, resting state 
default mode network activity, brain metabolism, and AD biomarkers. Forty overweight 
to obese women and men (between 55 and 70 years of age) will be randomized 1:1 into 
5-2 CR and control groups. In the 5-2 CR group, participants will be offered “healthy 
living” dietary counseling at baseline, which they will be instructed to implement for five 
days/week. For each of the other two consecutive days/week, they will consume two 
shakes (Boost ®, CWI Medical), providing a total of 480 Kcal/day. In the control group, 
participants will be offered “healthy living” dietary counseling at baseline, which they 
will be asked to implement for every day of the week. Participants will undergo 
screening including a history and physical examination, calculation of Body Mass Index 
(BMI, which must be ≥ 27) and a blood draw for insulin and glucose to determine 
whether they have insulin resistance. If participants meet eligibility criteria, they will 
continue with a baseline visit involving anthropometric measures, questionnaires, tests 
of cognitive function, brain MRI, blood draws for plasma and peripheral blood 
mononuclear cells, and lumbar puncture for cerebrospinal fluid biomarkers of AD. After 
8 weeks, we will collect the same measures. To assess and reinforce compliance with 
their respective diet, participants will come into the clinic every 2 weeks to discuss 
compliance, measure their body weight and perform blood draws for measurement of 
ketones to objectively confirm energy restriction for the 5-2 CR group.  We will also 
contact participants every week to further ensure compliance. To assess the effects of 
the diets on physical activity, participants will be asked to wear an accelerometer for 96 
hours before and after they are on the diet.  
 
 
 
 
 

 
 
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List of Abbreviations 
Abbreviation 
Definition 
5-2 CR 
5-2 Calorie Restriction 
Aβ1-42 
Amyloid-beta (1-42) peptide 
AD 
Alzheimer’s disease 
AE 
Adverse Event 
AGES 
Advanced Glycation End Products 
AL 
Ad libitum 
ALT 
Alanine Aminotransferase 
AOPP 
Advanced Oxidation Protein Products 
AST 
Aspartate transaminase 
ATP10A 
aminophospholipid translocase 
BDNF 
Brain Derived Neurotropic Factor 
BMI  
Body Mass Index 
BRC 
Biomedical Research Center 
CA3 
Cornus Ammoni 3 
CBC 
Complete blood count 
CMP 
Complete metabolic panel 
CSF 
Cerebral Spinal Fluid 
CVLT 
California Verbal Learning Test 
DMN 
Default Mode Network 
DNA 
Deoxyribonucleic acid 
FDA 
Food and Drug Administration 
fMRI 
Functional Magnetic Resonance Imaging 
FWA 
Federal Wide Assurance 
GABA 
gamma-Aminobutyric acid 
GIP 
Gastric Inhibitory Peptide 
GLP-1 
Glucagon Like Peptide-1 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HIPAA 
Health Insurance Portability and Accountability 
HEENT 
Head, eyes, ears, nose, throat 
HIV 
Human immunodeficiency virus 
HOMA-IR 
Homeostasis Model Assessment-Insulin Resistance 
IL-6, 8, 12, 23 
Interleukin-6, 6, 12, 23 
INR 
International normalized ratio 
IR 
Insulin Resistance 
IRB 
Institutional Review Board 
IV 
Intravenous 
Kg 
Kilogram 
LP 
Lumbar Puncture 
mL 
Milliliter 

 
 
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MMSE 
Mini Mental State Exam 
MMT 
Mixed Meal Test 
mRNA 
Messenger RNA 
MRS 
Magnetic Resonance Spectroscopy 
NAA 
N-Acetyl Aspartate 
NIA 
National Institute on Aging 
OGTT 
Oral Glucose Tolerance Test 
PANAS 
Positive and Negative Affect Schedule 
PBMC 
Periperial Blood Mononuclear cells 
pcASL 
Pseudo-continuous arterial spin labeling 
PSA 
Public service announcement 
PT 
Prothrombin time 
PTT 
Partial thromboplastin time 
QA 
Quality Assurance 
SAT 
Subcutaneous adipose tissue 
Si 
Insulin Sensitivity 
SOP 
Standard Operating Procedure 
TNF-α 
Tumor Necrosis Factor 
TSH 
Thyroid stimulating hormone 
UP 
Unanticipated Problem 
vmPFC 
Ventromedial prefrontal cortex 
WTI 
Wilms tumor protein 
 
 

 
 
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Table of Contents 
 
1. Introduction .......................................................................................................................... 8 
2. Study Objectives ............................................................................................................... 18 
2.a. Primary objectives ................................................................................................................. 19 
2.b. Secondary objectives ............................................................................................................. 20 
3. Subjects ................................................................................................................................ 20 
3.a. Description of study population ........................................................................................ 20 
3.b. Inclusion criteria .................................................................................................................... 20 
3.c. Exclusion criteria .................................................................................................................... 20 
4. Study Design and Methods ............................................................................................ 21 
4.a. Study overview ........................................................................................................................ 21 
4.b. Recruitment .............................................................................................................................. 23 
4.c. Screening ................................................................................................................................... 23 
4.d. Study procedures .................................................................................................................... 24 
4.e. End of participation ............................................................................................................... 32 
5. Management of Data and Samples .............................................................................. 32 
6. Additional Considerations ............................................................................................ 34 
6.a. Research with investigational drugs or devices ........................................................... 34 
6.b. Gene Therapy ........................................................................................................................... 34 
7. Risks and Discomforts .................................................................................................... 34 
8. Subject safety monitoring ............................................................................................. 38 
9. Outcome measures .......................................................................................................... 39 
9. a. Primary outcome measures ............................................................................................... 39 
9. b. Secondary outcome measures .......................................................................................... 39 
10. Statistical Analysis ........................................................................................................ 40 
10. a. Analysis of data/study outcomes ................................................................................... 40 
b. Power analysis ............................................................................................................................ 41 
11. Human subjects protection ........................................................................................ 44 
11. a. Subject selection .................................................................................................................. 44 
11. b. Justification for exclusion of children .......................................................................... 45 
11. c. Justification for exclusion of other vulnerable participants ................................. 45 
11. d. Justification of sensitive procedures ............................................................................ 46 
11. e. Safeguards for vulnerable populations ....................................................................... 46 
11. f. Qualifications of investigators ......................................................................................... 46 
12. Anticipated benefit ........................................................................................................ 47 
13. a. For adults ............................................................................................................................... 48 
b. For adults without consent capacity .................................................................................... 48 
13. c. For children ........................................................................................................................... 48 
13. d. Overall risk and benefit consideration ........................................................................ 48 
14. Consent Documents and Process .............................................................................. 48 
14. a. Designation of those obtaining consent ...................................................................... 48 

 
 
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14. b. Consent procedures ........................................................................................................... 49 
14. c. Consent documents ............................................................................................................. 49 
15. Data and Safety Monitoring ........................................................................................ 49 
15. a. Data and safety monitor .................................................................................................... 49 
15. b. Data and safety monitoring plan ................................................................................... 49 
15. c. Criteria for stopping the study or suspending enrollment or .............................. 49 
procedures ........................................................................................................................................ 49 
16. Quality assurance .......................................................................................................... 50 
16. a. Quality assurance monitor ............................................................................................... 50 
16. b. Quality assurance plan ...................................................................................................... 50 
17. Reporting of Unanticipated Problems, Adverse Events and Protocol ......... 50 
Deviations ............................................................................................................................... 50 
18. Alternatives to Participation ..................................................................................... 51 
19. Privacy ............................................................................................................................... 51 
20. Confidentiality ................................................................................................................ 51 
20. a. For research data and investigator medical records .............................................. 51 
20. b. For stored samples ............................................................................................................. 52 
c. Special precautions .................................................................................................................... 52 
21. Conflict of interest ......................................................................................................... 52 
21. a. Distribution of NIH guidelines ........................................................................................ 52 
21. b. Conflict of interest .............................................................................................................. 53 
21. c. Role of a commercial company or sponsor ................................................................. 53 
22. Technology transfer ..................................................................................................... 53 
23. Research and Travel Compensation ....................................................................... 53 
24. References ........................................................................................................................ 54 
25. Attachments/Appendices ........................................................................................... 61 
26. Consent forms ................................................................................................................. 68 
 
 
 
 

 
 
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1. Introduction 
 
Insulin signaling dysfunction and Alzheimer’s disease 
 
Type 2 diabetes increases the risk for developing sporadic Alzheimer’s disease (AD).1 
Insulin Resistance (IR) is defined broadly as reduced cellular responsiveness to insulin.2 
Peripheral IR, characterized by progressive hyperinsulinemia needed to maintain 
normoglycemia, may promote AD pathogenesis in the absence of hyperglycemia. For 
example, in the Rotterdam study, a two-fold increase in baseline insulin or in an index of 
IR over a 3-year period corresponded to a 40% greater likelihood of conversion to AD 
among participants without a history or present clinical diagnosis of type 2 diabetes.3  
 
In the brain, insulin normally facilitates microvascular blood flow, glucose uptake and 
metabolism,4 reduces accumulation and oligomerization of Aβ, 5-7 and inhibits 
phosphorylation of tau fibrils.8 Currently, there are no in vivo measures of brain IR, but it 
is assumed that it accompanies peripheral IR,9 which can easily be measured in vivo and 
studied. Peripheral IR has been associated with impaired glucose uptake in precuneus 
and prefrontal cortex10 and dysregulated brain insulin binding. Brain IR has recently 
been suggested to occur in AD even in the absence of peripheral IR,11 while AD-related 
pathology (Aβ-containing plaques and tau-containing tangles) and atrophy first occur in 
brain areas with a high density of insulin receptors that are also affected by early AD. 
These areas include the hippocampus, prefrontal cortex, cingulate cortices, and 
posteriomedial areas in parietal lobe such as the precuneus.12-14 Furthermore, 
peripheral IR, in rhesus macaques15,16 and humans,17-19 predicts neural atrophy in these 
regions. Finally, chronic insulin administration in rodents prevents atrophy in CA3 and 
neocortex in streptozotocin-induced diabetes.20 Thus, IR affects several physiological 
processes that are involved in neurodegeneration and AD etiopathogenesis.  
 
In terms of epidemiology, IR increases with age, lack of exercise, high calorie diets, and 
higher visceral adiposity.12,21,22 IR is present in many middle-aged23 and aged24,25 adults 
with a BMI above 27.26 Specifically, higher visceral adiposity due to being overweight or 
obese corresponds to higher IR in 25-40% of late middle-aged to geriatric Caucasian 
(Wilson and Kannel, 2002) and Asian27 populations. Overweight to obese participants 
(BMI ≥ 27) aged 55-70 years have insulin sensitivity values of approximately 7.6 
min−1/(μU mL−1) × 10−4.24,25 By extension, older overweight adults are at risk for 
developing AD12 and may show deficits in memory and executive function due to the 
effects of IR on the brain, as well as physiological changes associated with higher IR.  
 
CR improves insulin signaling and slows neurodegeneration in animals 
 
Most studies find that a restriction in caloric intake, without nutrient deficiency, extends 
lifespan and maintains optimal insulin signaling in mammalian species including rodents 
and non-human primates when the control animals are fed ad libitum and so develop 
age-related insulin resistance; CR eliminates aged non-human primates reduces or 

 
 
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eliminates the development of glucoregulatory impairment and cardiovascular 
disease.28-30 An important exception is recent work by Mattison and colleagues,31 who 
found that CR does not extend lifespan in the NIA CR rhesus macaque cohort, in which 
the monkeys in the control diet group were not overfed and not obese. Interestingly, 
monkeys who began CR in middle age (13-17 years) had significantly less 
glucoregulatory dysfunction versus controls, but not monkeys who began CR as 
juveniles. This NIA study underscores the importance of studying CR effects on middle 
age. Furthermore, long-term 30% CR relative to baseline intake increased insulin 
sensitivity in the Wisconsin National Primate Research Center monkey cohort, which 
began CR exclusively in middle age. Regarding the brain, this increased sensitivity was 
associated with improved cognition relative to controls, as well as more gray matter 
volume in hippocampus, prefrontal cortices, and other areas affected by early AD.16  
 
Recent findings from animal studies suggest that alternate day fasting can have similar 
beneficial effects, even with relatively modest 10-25% reductions in overall calorie 
intake.32-34 The brain effects of CR may be due to several biological mechanisms 
impacted by improved insulin signaling, such as: 1) increased brain-derived neurotrophic 
factor (BDNF), which is significantly lower after diet-induced IR in mice; 2) decreased 
pro-inflammatory cytokine levels; 3) decreased production of reactive oxygen species 
(ROS) that damage macromolecules; and 4) improved energy metabolism manifested by 
increased insulin sensitivity and changes in leptin, adiponectin, and other 
hormones.32,33,35-38 We found that an alternate day CR regimen over 8 weeks, in 
overweight asthma patients, reduced peripheral IR and plasma inflammatory markers.39  
 
A study comparing continuous restriction and 5-2 CR in overweight women, defined as 2 
consecutive days of consuming 500-600 kilocalories followed by non-restricted eating 
for 5 days, indicated that both regimens produced comparable effects to alternate day 
restriction.40 Both continuous CR and 5-2 CR were tailored to result in a net 20% energy 
restriction. Critically, 5-2 CR led to significantly higher insulin sensitivity relative to 
continuous restriction and was easier for participants to maintain. We previously 
showed that 8 weeks of alternate day CR induces multiple effects of interest,39 such as 
decreased weight (~8% of baseline body weight), decreased TNF-α (Figure 1), and 
decreased levels of ROS-induced 8-isoprostane, protein carbonyl, and nitrotyrosine 
(Figure 2).  
 

 
 
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Figure 1. The effect of alternate day calorie restriction on serum TNF-α. By the 8-week 
time point, concentrations were greatly reduced regardless of assessment on ad libitum 
(AL) or restricted (CR) days. 
 
 
Figure 2. The effect of alternate day calorie restriction on serum levels of nitrotyrosine.  
By the 8-week time point levels decreased regardless of assessment on ad libitum (AL) 
or restricted (CR) days. 
 
Study aims and overview 
 
This study is a proof-of-concept exploratory pilot study, which will generate several 
kinds of preliminary data to guide further studies in the field. Using a randomized 
clinical trial design, we will compare the effects of 5-2 CR and a control diet. We will 
compare various outcome measures between groups, but also to baseline on a within-
groups basis.  
 
The following main outcomes will be assessed: 1) The ratio of Tyr and Ser 
phosphorylated forms (p-panY and pSer312) of insulin receptor substrate 1 (IRS-1) in 
plasma exosomes enriched for neuronal origin, which is a novel biomarker of brain IR 
(primary) (44); 2) peripheral insulin sensitivity derived from a mixed meal tolerance test; 

 
 
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3) brain fMRI activation during presentation of appetitive “junk” foods versus non-
appetitive foods; 4) brain fMRI activation during rest; 5) brain metabolism measured by 
magnetic resonance spectroscopy (MRS); and 6) cognitive performance on memory and 
executive function tasks. Due to the exploratory nature of this study, we will also collect 
many secondary outcomes that inform on the relevant mechanisms for our primary 
outcome of interest, or are biomarkers of AD. Secondary measures include but are not 
limited to anthropometric measures of obesity (body weight, waist circumference, BMI, 
and sagittal diameter, which reflects visceral fat particularly well41), physical activity 
measured by monitoring devices, blood and CSF biomarkers relevant to AD, and other 
parameters detailed below.  
 
Study population: We will study women and men aged 55-70 years with BMI ≥ 27 and 
confirmed peripheral IR at baseline. Peripheral IR is mostly present in middle-aged23 and 
aged24,25 adults with a BMI above 27, even in the absence of type 2 diabetes. 
Specifically, higher visceral adiposity due to being overweight or obese corresponds to 
higher IR in 25-40% of late middle-aged to geriatric Caucasian42 and Asian27 populations. 
Overweight to obese participants (BMI ≥ 27) aged 55-70 years have insulin sensitivity 
values of approximately 7.6 min−1/(μU mL−1) × 10−4.24,25 We will study participants aged 
55-70 years to examine brain IR and other AD-associated biomarkers and measures 
during late middle-age/early old age, which temporally correspond to the preclinical 
stage of AD. Late middle-life/early old age overweight adults are at higher risk for 
developing AD12 and may show mild deficits in executive function due to the effects of 
IR on the brain, as well as physiological changes associated with higher IR.  
 
We will exclude subjects with stroke and other neurological diseases of the central 
nervous system that may impact structural integrity, metabolism and function in 
affected brain regions and confound the results of MRI, MRS, as well as CSF and 
exosomal biomarkers. We will exclude subjects that abuse illicit drugs since these may 
impact brain metabolism and function and may confound the effects of the two diets on 
brain biomarkers. We will exclude subjects with clinically significant endocrine disorders 
such as hypothyroidism, Cushing’s Syndrome, etc. can alter peripheral and brain 
metabolism, body weight, and appetite and may confound the effects of the two diets 
on relevant outcome measures. We will exclude subjects with history of anorexia 
nervosa or bulimia, since these disorders may complicate compliance with the two diets 
or raise concerns for the safety of implementing a new dietary pattern. In addition, 
subjects with Type 2 diabetes, taking anti-diabetic medications or with history of 
hypoglycemia may not be able to safely engage in 5-2 CR, due to the possibility of 
developing symptomatic hypoglycemia. We will exclude subjects with chronic HIV, HCV, 
or HBV infections, since these are associated with metabolic and endocrine 
abnormalities and may confound the effects of the two diets. We will exclude subjects 
with low hematocrit, since it might suggest an undiagnosed underlying condition and 
limit our ability to obtain blood samples at various times during the study. We will 
exclude subjects with elevated AST and/or ALT > 1.5 times the upper normal limit 
suggesting significant liver disease, which may have an impact on the effects of the two 

 
 
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diets. We will exclude subjects receiving systemic corticosteroids that can induce IR and 
alter multiple aspects of metabolism and alter the effects of the two diets. Given the 
profound effects of APOE genotype on AD biofluid and imaging biomarkers and AD risk 
in midlife,43-46 it is customary for studies of AD biomarkers and cognition in preclinical 
cohorts, to control for APOE-associated variance as a covariate.19,47 In addition, there is 
evidence for a complex interaction of APOE, sex and IR regarding their effects in AD.48 
Therefore, APOE genotyping will be performed for all participants.  
 
Brain IR (primary outcome): We have recently developed a methodology for exosome 
isolation from stored plasma samples and enrichment for neuronal-origin based on 
immunoreactivity towards neuronal cell adhesion molecules (NCAM and/or L1)49 
expressed on the surface of a sub-population of these exosomes. First, we implemented 
this methodology in a cross-sectional study measuring AD pathogenic proteins (Aβ42, 
total Tau, p-T181-Tau, p-S396-Tau) in L1/NCAM expressing exosomes isolated from 
blood samples of 54 AD patients and 54 cognitively normal age- and sex- matched 
controls. Exosomal Aβ42, p-T181-Tau, and p-S396-Tau were significantly higher in AD 
patients compared to controls achieving near-perfect discrimination between patients 
and controls with an ROC AUC ~ 1.50 Subsequently, we performed a study looking for 
biomarkers of brain IR in 26 patients with AD, 26 cognitively normal age- and sex-
matched controls without diabetes and 20 age- and sex-matched controls with diabetes. 
We measured two phosphorylated forms of IRS-1 that differ in terms of downstream 
insulin signaling, one relatively active (p-panY-IRS-1) and one relatively inactive (p-S312-
IRS-1); the ratio of the two forms in a tissue is a measure of insulin resistance.  We 
found that AD patients had significantly lower p-panY-IRS-1 and higher p-S312-IRS-1 
compared to both diabetic and non-diabetic controls, while diabetic controls had 
intermediate values between AD patients and non-diabetic controls.51 These findings 
suggest the presence of brain selective IR in patients with AD and, to a lesser extent, in 
individuals with diabetes.  In addition, in a longitudinal series of 24 patients, with 
samples 1 - 10 years prior to diagnosis of AD or amnestic MCI and at the time of 
diagnosis, and 24 age- and sex- matched controls, we found that p-panY-IRS-1, p-S312-
IRS-1, and their ratio, are abnormal 1-10 years prior to AD/aMCI diagnosis (Figure 3). 
Therefore, brain IR is present at the preclinical stage of AD and these brain IR exosomal 
biomarkers may be used to predict AD at the preclinical stage as early as 10 years prior 
to diagnosis.51 

 
 
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Figure 3. Mean exosomal pS312-IRS-1/p-panY-IRS-1 in neuronal enriched exosomes in 
22 patients with AD at the time of first diagnosis and 1-10 years preclinically, as well as 
in cognitively normal age and sex-matched controls.51 
 
In the present study, we hypothesize that brain IR biomarkers will respond to CR and 
that the 5-2 CR group will show increased p-panY-IRS-1, decreased p-S963-IRS-1, and 
increased ratio, compared to baseline and to controls. 
 
Peripheral Insulin Sensitivity: HOMA-IR and peripheral insulin sensitivity (SI) will be 
used to assess peripheral IR. HOMA-IR is calculated from fasting glucose and insulin. 
HOMA-IR strongly corresponds to insulin sensitivity determined from euglycemic or 
hyperinsulinemic clamp.52 HOMA-IR will primarily be used to screen participants to see 
if they have peripheral insulin resistance (see below). SI will be derived from a mixed 
meal tolerance test. The typical SI measurement is derived from a 75 g oral glucose 
tolerance test (OGTT) and shows high correspondence with the “gold standard” 
euglycemic-hyperinsulinemic clamp method.53 The mixed meal has the advantages of 
enabling the assessment of incretins such as glucagon-like peptide-1 (GLP-1) and gastric 
inhibitory peptide (GIP), and acting as a more natural stimulus of islet beta cells that 
secrete insulin.54 Lower SI corresponds to higher IR. For defining a cut-off inclusion 
criterion for this study, we consider an HOMA-IR value greater than 1.8 as evidence of IR 
in overweight, late middle-aged to aged participants without type 2 diabetes,52 whereas 
median HOMA-IR was 2.89 among participants with type 2 diabetes in the Matthews et 
al. study. We hypothesize that the 5-2 CR group will show increased SI compared to 
baseline and to controls.  
 
In addition, we will assess the effects of the two diets on these related secondary 

 
 
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measures: anthropometric measures of obesity, such as body weight, BMI, waist 
circumference, sagittal diameter41; and clinical laboratory measures reflecting 
peripheral metabolism and glucoregulatory control, such as plasma lipids and HbA1c.  
 
Neuroimaging - Brain function: We will use fMRI to probe brain activation during a food 
rating task concerning appetitive “junk” foods versus non-appetitive foods. Moderate 
CR in humans typically does not affect appetite.39,55 Instead, restriction decreases food 
cravings for and consumption frequency of energy-dense foods during CR55,56 and 
several months after resumption of regular diet.29 The neural correlates of this change 
in behavioral responsiveness have not been investigated.  We hypothesize that viewing 
images of energy-dense foods activates meso-cortico-limbic regions involved in reward, 
like striatum, prefrontal cortex, insula, and ventral tegmental area57; deactivation of 
these regions has been seen with voluntary cognitive reappraisal.58 We hypothesize that 
participants on 8 weeks of 5-2 CR will show decreased liking ratings for energy-dense 
foods and corresponding decreased fMRI activation compared to controls.  The fMRI 
paradigm we will use was developed by Antonio Rangel at the California Institute of 
Technology and has been used in multiple published studies (such as Harris et al., 
Temporally dissociable mechanisms of self-control: early attentional filtering versus late 
value modulation, J Neuroscience 2013; Hare et al., Focusing attention on the health 
aspects of foods changes value signals in vmPFC and improves dietary choice, J 
Neuroscience 2011; etc) to probe perceived value and salience of food stimuli that drive 
dietary choices, as well as mechanisms of self control. 
 
We will also assess the effects of the two diets on intrinsic functional connectivity of the 
default mode network (DMN), a major and one of the earliest targets of AD pathology.59 
IR is related to decreased glucose uptake in the precuneus, the key node of the DMN, in 
cognitively normal older adults with pre-diabetes.10 Conversely, acute intranasal insulin 
administration in participants with type 2 diabetes increases DMN activity, suggesting 
that insulin modulates DMN neural metabolism and firing. The levels of CSF AD 
biomarkers (Aβ1-42, total tau, p181Tau) in cognitively intact adults are also associated with 
changes in DMN connectivity.60 We hypothesize that participants on 8 weeks of 5-2 CR 
will improve DMN connectivity compared to controls eating normally for 8 weeks. 
 
Finally, as a secondary measure, we will assess the effects of the two diets on brain 
activity underlying executive function performance. This task is exploratory because no 
existing studies examine the effect of IR or IR-targeted therapies on Stroop-induced 
brain activity. To this end, we will perform fMRI during a Stroop Color-Word task, a 
standard test of executive function.61 The Stroop effect62 in this paradigm consists of 
reading a color word (e.g. the word “red”) and making a selection of either the color 
word or the font color. The font color can be either congruent (the word “red” is colored 
red) or incongruent (e.g. the word “red” is colored yellow) to the color word. Inhibitory 
control is needed to disregard the incongruent color word and select the font color. This 
Stroop effect produces robust activation in prefrontal cortex,61 a target area of interest, 
since its activity is negatively associated with IR.19 We hypothesize that participants on 8 

 
 
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weeks of 5-2 CR will show improved performance on Stroop in association with 
decreased activation (suggesting lesser effort) compared to controls. 
 
Neuroimaging - Brain metabolism: To assess the effects of the two diets on brain 
energy metabolism we will use magnetic resonance spectroscopy (MRS), a technique 
used to measure chemical concentrations in the brain. Specifically, within regions of 
interest, we will measure concentrations of N-Acetyl Aspartate (NAA), glucose, and 
lactate normalized to creatine to assess: 1) neural density (NAA), 2) glucose metabolism, 
reflected by brain glucose concentration reflecting the balance between glucose 
delivery through the blood brain barrier and consumption, and 3) metabolic reliance on 
glycolysis (lactate). These metabolites will be measured in the hippocampus and 
prefrontal cortex, areas preferentially vulnerable to AD pathology that show impaired 
glucose uptake as IR increases.10 We will also derive concentrations of additional 
metabolites, such as neurotransmitters like glutamate and GABA,63 which are decreased 
in AD as a result of impaired brain metabolism.64 We hypothesize that, by improving 
brain metabolism, participants on 8 weeks of 5-2 CR will show increased concentrations 
of glutamate and GABA and decreased glucose (due to increased utilization) and lactate 
(due to decreased production) compared to controls.  
 
Cognition and affect: Moderate CR has profoundly beneficial effects on memory, such 
as the California Verbal Learning Test (CVLT), in sedentary aged adults at risk for AD,65 
an effect that may be associated with insulin resistance. Consequently, we will assess 
verbal memory for unstructured information with the CVLT, as well as structured 
information with Logical Memory I and II; these tests are common neuropsychological 
measures. Memory recall generally begins to decline starting in the fifth decade of life,66 
while peripheral IR may be related to subclinical declines in memory performance within 
the range of normal variability, but also subclinical hippocampal microstructural 
changes.67,68 In addition, higher IR in older adults predicts executive function deficits in 
Trails A and B 69, as well as the Symbol Digit Modalities Test and Stroop Color-Naming 
task.70 We intend to measure executive function using the newly developed 
computerized assessment tool called “Examiner” (http://examiner.ucsf.edu/).  This 
software reliably and validly assesses domains of executive function and is intended to 
become the “gold standard” for clinical trials. We hypothesize that participants on 8 
weeks of 5-2 CR will show better performance on executive function and memory tasks 
compared to baseline and controls, within the normal range. 
 
Regarding secondary measures specific to cognition, we screen out subjects with 
subclinical cognitive dysfunction by using the Mini Mental State Exam (MMSE) and an 
inclusive cut-off of 26 out of 30. Given the possibility of psychological stress associated 
with the two diets, we will gauge mood using the Positive and Negative Affect Schedule, 
or PANAS71 and the general quality of life of participants using the RAND Health 36-Item 
Short Form Health Survey (RAND SF-36) as previously described.40 These questionnaires 
use Likert scales that assess mood, level of energy, difficulty concentrating, headaches, 
feeling cold, constipation, specific negative emotions (depression, anxiety, and 

 
 
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irritability), hunger and preoccupation with food. We hypothesize that participants on 8 
weeks of 5-2 CR will show improved positive over negative affect and general quality of 
life compared to controls. 
 
AD biofluid biomarkers: To assess the effect of 5-2 CR on AD pathogenesis, we will 
assess additional plasma exosomal protein and mRNA exosomal biomarkers of AD.50 In 
addition, we will assess traditional AD biomarkers in CSF: Aβ1-42, total tau, p181Tau, and 
soluble APP fragments. Extracellular Aβ oligomerization resulting in decreased CSF Aβ1-42 
is thought to begin during the preclinical phase of AD, perhaps decades before clinical 
disease onset. Even mild glucoregulatory dysfunction in older adults diagnosed with AD 
was associated with greater Aβ burden.72 By extension, IR in late middle age may also be 
related to Aβ burden. Intracellular tau deposition (and increased CSF concentration) 
increases during preclinical and clinical disease, in concert with neurodegeneration and 
cognitive deficits.73 For tau protein, insulin resistance dysregulates phosphorylation of 
the β subunit.74 We hypothesize that participants on 8 weeks of 5-2 CR may show 
increased CSF Aβ1-42, no change in total tau, but decreased p181Tau compared to 
controls.  
 
In addition, we also intend to assess a number of less well-established or novel AD 
biomarkers, which reflect various aspects of AD etiopathogenesis. These include 
chemicals in CSF, plasma, serum and whole blood, such as neurotrophic factors, 
cytokines and inflammatory factors and cells, various peptides, proteins, lipids, and 
RNAs. In terms of neurotrophic factors, BDNF is an important pleiotropic factor that 
promotes synaptic plasticity and protects against the cytotoxic effects of Aβ, where AD 
patients illustrate worse cognitive performance in association with lower CSF BDNF 
levels.75 Pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α 
(TNF-α) and IL-12 are released by microglia and predict elevated Aβ levels in 
transgenic76,77 and wild-type rodents,78 as well as reduced synaptic plasticity79 and other 
aspects of neurodegeneration. Increased production of reactive oxygen species 
mediates neural atrophy by damaging mitochondrial DNA and degrading 
macromolecules such as proteins and sugars,80 leading to advanced gylcation end 
products (AGEs) and advanced oxidation protein products (AOPP) formation and further 
cellular damage. Importantly, a previous 8-week intermittent CR study39 showed that 
markers of inflammation and oxidative stress in blood were significantly reduced 
(Figures 1 and 2).  
 
Finally, we will measure proteins and RNAs implicated in AD pathogenesis and insulin 
signaling in neuronal-origin plasma exosomes.  These include, but are not limited to Aβ1-
42, total tau, p181Tau, p-S396-Tau, p-S312-IRS-1, p-panY-IRS-1, cathepsin-D, ubiquitin, 
LAMP2, HSP70, and CD81.81 
 
Physical activity: To assess baseline activity-associated variance in other outcomes, as 
well as the effects of the two diets on physical activity, we will use accelerometry. We 
hypothesize that the 5-2 CR group will not show any differences in physical activity 

 
 
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compared to baseline and the control group. Nonetheless, this analysis is expected in 
many manuscripts on dietary interventions, in order to rule out effects attributed to 
differences in physical activity. 
 
Gene expression: To understand the molecular mechanisms of the benefits of calorie 
restriction, several studies have utilized gene expression changes using microarray in 
mammals. In a recent meta-analysis of publically available CR microarray data from 
mouse, rat and pigs in gene expression changes were analyzed across various tissues 
including liver, kidney, heart, mammary glands, muscle, lung, brain, and adipose.82 
There were 174 genes that were significantly over- or under-expressed, some of which 
were tissue-specific but others were found in more than 3 tissue types. The functional 
categories that were enriched included genes involved in growth hormone signaling, 
lipid metabolism, immune response, retinol metabolism, copper ion detoxification, and 
circadian rhythms. These results highlighted the complex patterns of changes that occur 
with CR that span across many domains.  
 
A handful of studies have examined the gene expression changes that occur with caloric 
restriction in humans, particularly in fat cells. In a study of 36 healthy young (mean age 
~36.8 + 10 years old) overweight individuals, randomized to control, 25% CR and 12.5% 
caloric restriction with 12.5% increase energy expenditure (CREX) the changes in 
expression of mitochondrial biogenesis genes in skeletal muscle cells was tested.83 
Compared to control group, subjects in CR and CREX group showed increase expression 
of mitochondrial genes (PPARGC1A, TFAM, eNOS, SIRT1, and PARL).  In particular, 
expression of SIRT1 was increased by 113% in CR and 61% in CREX group.  In a study of 
3-month CR study in 22 obese women, genome-wide expression analysis in 
subcutaneous adipose tissue (SAT) showed a down-regulation of 474 adipocyte genes 
and up-regulation of 511 macrophage-related inflammation genes.84 In a second study, 
7 low- and high- weight loss responders to a 6-month hypo-caloric diet were assessed 
for genome-wide expression differences SAT.85 While no differences in gene expression 
were observed before the intervention, 644 genes were differentially expressed after 
the dietary intervention. Pathway analysis of differentially expressed genes suggests an 
enrichment of genes involved in angiogenesis and cerebellum function. Interestingly, 
up-regulation of eNOS observed in muscle cells was also observed in SAT. It is unclear 
whether there were other overlaps between SAT and skeletal muscle in response to CR.  
In a slightly different study design, the final study looked at gene expression differences 
for ~8500 genes in SAT between women who were successful and unsuccessful at 
weight-loss following 6-month dietary intervention on one of 4 standard diets that 
varied in glycemic index and macronutrient composition following an 8-week period of 
CR.86 The global expression pattern was able to discriminate between responders 
(N=22) and non-responders (N=22) to the weight loss protocols. These studies provide 
promising evidence for the feasibility to detect changes in gene expression under 
different dietary conditions in humans. 
 
We expect to find a unique gene expression signature in whole blood after 5-2 CR. We 

 
 
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anticipate that several expression changes in domains similar to mammalian studies will 
be observed in humans including genes that encode proteins involved in growth 
hormone signaling, lipid metabolism, and immune response.  We will take two 
approaches to analyze the expression data. First expression changes in genes previously 
implicated in CR will be tested. These include genes involved in mitochondrial function, 
insulin signaling, BDNF pathways, autophagy, mTOR pathways, DNA repair, antioxidant 
system and cell survival. Second, an unbiased approach will be taken by analyzing 
genome-wide expression changes.  
 
Methylation in Leukocytes: There are two studies that have looked at genome-wide 
methylation in SAT and peripheral blood mononuclear cells (PBMCs) in humans.85,87 In 
the study of DNA methylation in SAT following 6-month CR, differences at 35 loci were 
found between the high and low weight loss responders at baseline suggesting 
methylation signature can be predictive of those who will respond to CR intervention.  
Three of these regions showed differential methylation after CR.85 Not surprisingly, 
differential methylation was observed in genomic regions that contain genes linked with 
weight control and insulin regulation including the cholecystokinin B receptor gene.  In 
the second study, differential methylation in PBMC was studied in 12 subjects who were 
either low- or high- weight loss responders to 8-weeks of caloric restriction.87 At 
baseline, several CpG located in the aminophospholipid translocase (ATP10A) and cell 
surface glycoprotein 44 (CD44) genes differed between the two groups suggesting that 
methylation patterns in this region are important for weight loss in response to CR. At 
the end of the study, methylation differences were observed at the promoter region of 
Wilms tumor protein (WT1). Interestingly, the methylation at ATP10A and WT1 
significant changed during the 8-week intervention. The biological relevance of these 
genes with caloric restriction is unknown. One of the limitations of the study reported 
by Milagro and colleagues is that their analyses did not account for white blood cell 
differential counts. This is particularly concerning when studying weight loss since 
changes in leukocytes have been documented and shown to explain 10% of the variance 
in weight loss over 2 years.88 Therefore, in our study, we intend to measure white blood 
cell and lymphocyte differential counts at the times of PBMCs acquisition. We expect to 
find a unique methylation signature with 5-2 CR in PBMCs.  As with the expression data, 
we will use a two-pronged approach for the analysis of methylation data where we take 
a targeted candidate pathway approach and an agnostic genome-wide methylation 
change approach. Furthermore, our study may replicate some of the PBMC DNA 
methylation changes observed in CR findings reported by Milagro.87 
 
Future studies: Finally, CSF and plasma samples will be saved for future studies on the 
effects of the two diets on metabolism (e.g. hormones of metabolism, such as incretins, 
glucagon, etc.), proteomic and metabolomic studies.  
 
2. Study Objectives 
 

 
 
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Hypotheses and Specific Aims 
 
1. To assess the effects of the two diets on exosomal p-S312-IRS-1, p-panY-IRS-1, and 
their ratio (brain IR);  
2. To assess the effects of the two diets on other exosomal protein and mRNA 
biomarkers of AD; 
3. To assess the effects of the two diets on SI and HOMA-IR (peripheral IR);  
4. To assess the effects of the two diets on liking ratings for calorie-rich foods and 
associated fMRI activation versus non-appetitive foods; 
5. To assess the effects of the two diets on Stroop-associated fMRI activation; 
6. To assess the effects of the two diets on intrinsic functional connectivity of the 
(DMN) assessed by resting fMRI; 
7. To assess the effects of the two diets on glutamate, GABA, glucose and lactate in 
prefrontal cortex and hippocampus; 
8. To assess the effects of the two diets on memory and executive function; 
9. To assess the effects of the two diets on mood, level of energy, difficulty 
concentrating, headaches, feeling cold, constipation, negative emotions (depression, 
anxiety, irritability), hunger and preoccupation with food, and quality of life; To 
assess the effects of the two diets on clinical Labs: lipid panel, uric acid; 
10. To assess the effects of the two diets on plasma ketones, such as β-hydroxybutyrate 
and other metabolites; 
11. To assess the effects of the two diets on plasma and CSF AD biomarkers, including 
Aβ1-42and p181Tau; 
12. To assess the effects of the two diets on plasma and CSF neurotrophic factors, such 
as BDNF; 
13. To assess the effects of the two diets on plasma and CSF reactive oxygen species,  
advanced glycation end-products and advanced oxidation protein products; 
14. To assess the effects of the two diets on plasma and CSF pro-inflammatory 
cytokines, such as IL-6, IL-8, IL-12 and IL-23; 
15. To assess the effects of the two diets on anthropometric measures (weight, waist 
circumference, sagittal diameter); 
16. To assess the effects of the two diets on physical activity as measured by 
accelerometry; 
17. To assess the effects of the two diets on expression pattern of genes in candidate 
pathways; 
18. To assess the effects of the two diets on the global gene expression pattern in 
PBMCs; 
19. To assess the effects of the two diets on methylation patterns in genes of candidate 
CR pathways; 
20. To assess the effects of the two diets on global methylation patterns in PBMCs. 
 
2.a. Primary objectives 
 
Primary objective is to demonstrate that 8-weeks of 5-2 CR plus “healthy living” 

 
 
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counseling in a group of overweight, late middle-aged to aged participants will 
significantly increase the ratio of plasma exosomal p-panY-IRS-1 to p-S312-IRS-1, 
compared to baseline and to controls who only receive “healthy living” counseling.  
 
2.b. Secondary objectives 
 
Secondary objectives are noted under specific aims 5-21. Collectively, we aim to 
demonstrate that 8-weeks of 5-2 CR plus “healthy living” counseling in a group of 
overweight, late middle-aged to aged participants will significantly improve peripheral 
insulin sensitivity, decrease fMRI activation in meso-cortico-limbic areas involved in 
appetitive food salience and reward pathways, increase intrinsic functional connectivity 
of the DMN, improve brain metabolism, AD biomarker profile and cognitive 
performance compared to baseline and controls who only receive “healthy living” 
counseling. 
 
3. Subjects 
 
3.a. Description of study population 
 
The study population will consist of overweight, older (55-70 years old), men and 
women with evidence of IR and central obesity. The requirement for IR was set because 
it defines a population at increased risk for AD,89 and IR may be responsive to brief39 or 
extended90 CR in overweight human participants. The upper and lower age limits were 
set because we are interested in the effects of IR and of 5-2 CR on the preclinical phase 
of AD, at midlife, 15-20 years prior to the onset of clinical disease.  
 
The accrual target is 100 participants to allow for 40 participants that will be successfully 
enrolled and complete the study and 60 screen failures and withdrawals. Withdrawals 
will be replaced.  
 
3.b. Inclusion criteria 
 
1. BMI ≥ 27; in addition, weight £ 350 lbs (weight limit for MRI scanner); 
2. Age of 55-70 years; 
3. HOMA-IR ≥ 1.8;  
4. MMSE ≥ 26 
 
3.c. Exclusion criteria 
 
1. History of cardiovascular disease; 
2. History of clinically significant stroke or other neurological disease of the central 
nervous system; 
3. History of substance abuse in the past 6 months or positive urine drug screen; 
4. History of clinically significant endocrine disorders (common mild endocrine 

 
 
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disorders, such as untreated subclinical hypothyroidism with TSH < 10 mU/l or 
successfully treated hypothyroidism may be allowed); 
5. History of eating disorders, significant GI disorders or malabsorption disorders; 
6. History of type 2 diabetes; and/or use of anti-diabetes medications or insulin; and/or 
type 2 diabetes diagnosed during the screening visit based on fasting glucose > 125 
mg/dL;  
7. History of hypoglycemia; and/or a fasting glucose < 70 mg/dL during the screening 
visit. 
8. Current use of systemic corticosteroids; 
9. Positive screening tests for HIV, HCV or HBV; 
10.  Hematocrit less than 35% or hemoglobin less than 11 mg/dL; 
11.  ALT or AST > 1.5 times the upper normal limit; 
12.  Contraindications for MRI (pacemakers, ferrous metal implants or shrapnel in or 
around the head, etc.). 
13. Contraindications to LP, such as Coumadin, coagulopathy (international normalized 
ratio, or INR > 1.5; prothrombin time (PT), partial prothrombin time (PTT) > 1.5 x 
upper normal limit). Aspirin 81 mg qd is allowed. Aspirin up to 325 mg qd is allowed, 
if withheld for 7 days prior to the LP. 
14. Pregnancy or nursing. 
15. Refusal to consent to genetic testing for APOE. 
 
4. Study Design and Methods 
 
4.a. Study overview 
 
This study is a randomized clinical trial, where participants will be assigned into two 
groups.  In the 5-2 CR group, participants will be offered “healthy living” dietary 
counseling at baseline, consisting exclusively of published literature from the CDC and 
USDA, which they will be instructed to implement for five days/week. In addition, they 
will be instructed to consume only two shakes (Boost®, CWI Medical) providing a total of 
480 Kcal/day for two consecutive days/week. In the control group, participants will be 
offered “healthy living” dietary counseling at baseline, which they will be instructed to 
implement for every day of the week.  
 
Should a participant not be able to keep their scheduled study visits due to an 
unforeseen circumstance, and they are on 5-2 CR, she or he may be asked to implement 
CR on another day, or to extend the 5-2 CR diet for an additional week. Voluntary 
dropouts will be replaced until we have acquired complete data from 40 participants.  
 
4.a.1. Experimental conditions: Both groups will receive “healthy living” brochures 
published by the CDC and USDA at baseline. In the 5-2 CR group, participants will be 
encouraged to implement “healthy living” diet for five days/week, and not to 
intentionally overeat. For 2 consecutive days per week, participants in the 5-2 CR group 
will consume 480 kilocalories in the form of two meal replacement shakes (Boost®, CWI 

 
 
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Medical). These shakes will be supplied at the end of the Baseline Visit, as well as during 
the Compliance Visits every 2 weeks. This dietary regimen achieves about 20% net 
restriction compared to their weekly caloric intake, assuming a baseline 2000 Kcal/day 
intake. Participants in the 5-2 CR group will have the opportunity to taste the meal 
replacement shake (Boost®, CWI Medical) at the end of the Baseline Visit and choose a 
flavor they prefer. In the control group, participants will be encouraged to follow the 
“healthy living” diet every day.  
 
Rationale for control condition: We wish to implement a low intensity form of dietary 
intervention as an active placebo, by providing published material on “healthy living” 
diet to both groups. In general, counseling has very modest effects on body weight, if 
any. For example, Noda and colleagues91 examined weight loss over 8 weeks among 
participants who initially received 30-60 minutes of dietary counseling (n=44) vs. no 
counseling (n=47). Whereas the dietary counseling group showed modest weight loss (-
0.6 kg) compared to baseline and weight in the no counseling group did not change (0 
kg), there was no overall significant effect of Intervention Group. Six months of intensive 
dietary counseling, defined as weekly consultation for the first month and biweekly 
thereafter, led to modest weight loss (-2.5 kg)92 – in this study, we will implement a 
much less intensive form of dietary counseling. Therefore, dietary counseling alone is 
not an effective way for achieving weight loss. By contrast, Harvie et al.40 found that 8 
weeks of 5-2 CR led to a reduction of 6.5 kg. In addition to providing an active placebo, 
implementing the same dietary counseling to both groups, we intend to make the 5-2 
CR group’s diet during non-CR days less variable and similar to that of the “Healthy 
living” group. Finally, by offering an intervention to control subjects, we intend to 
increase their motivation for completing the study. Counseling is routinely used as a 
control in studies implementing behavioral interventions, such as diet and exercise.93  
 
Although, we do not wish to implement detailed dietary counseling besides providing 
subjects with published material and reviewing it with them, the study Investigators 
have received advice by long-time collaborator at the US Department of Agriculture’s 
Beltsville Human Nutrition Research Center Dr. David Baer. The Center is specialized in 
testing novel dietary interventions and implementing comprehensive behavioral 
interventions to ensure compliance. Study investigators and the Clinical Research 
Coordinator have become acquainted with their methods and will continue to consult 
with them throughout this study whenever applicable. The Clinical Research 
Coordinator or the PI will review the informational materials with participants and 
provide any necessary clarifications. 
 
4.a.2. Compliance: We will assess and reinforce diet compliance with correspondence 
by telephone or electronic mail on week 1, 3, 5 and 7, as well as with Compliance Visits 
on weeks 2, 4, and 6. To assess compliance during Compliance Visits, we will be 
measuring plasma β-hydroxybutyrate. Values around 2-2.5 μM/mL during CR days 
suggests compliance during 5-2 CR. β-hydroxybutyrate values of 0.5-1.0 μM/mL suggest 
non-compliance with 5-2 CR, but would be expected for non restricted days and for the 

 
 
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control group.39 In addition, we will be measuring body weight. Weight loss for 5-2 CR is 
expected to be about 2% per week based on our previous work.39 Therefore, a 
progressive decrease seen after 2 weeks of 5-2 CR and persisting in the 5-2 CR group 
over 8 weeks suggest compliance to 5-2 CR,39 while a smaller decrease in body weight is 
expected for the control group. 
 
In addition, we will contact participants by telephone or electronic mail (using the 
Secure Email & File Transfer Service: https://secureemail.nih.gov/bds/Login.do) weekly 
to assess not only compliance with the diets, but also mood, level of energy, difficulty 
concentrating, headaches, feeling cold, constipation, negative emotions 
(sadness/depression, anxiety, irritability), hunger and preoccupation with food.39,40,94 
For e-mail communications, we will provide participants with the web-address of the 
Secure Email & File Transfer Service with instructions on how to acquire login 
credentials. 
 
4.b. Recruitment 
 
Participants may be recruited through direct advertisement. Brochures and flyers will be 
used. Recruitment letters may be mailed to health care providers and to those 
participants who have requested information. We may place ads and/or public service 
announcements (PSA) on websites, in radio broadcasts, television, as well as 
newspapers, newsletters, and magazines. Direct mailing through established marketing 
and advertising organizations might also be used. Pre-screening over the telephone will 
utilize the eligibility checklist. Participants that we believe are likely to be eligible will 
come to the NIA-Clinical Research Unit to undergo screening. 
 
We anticipate an accrual rate of 2-6 participants per month. 
 
4.c. Screening 
 
Subjects will arrive on the NIA-Clinical Research Unit after a 12-hour fast from the 
previous night. Informed consent will be obtained from the subject prior to any 
procedures. The Screening Visit will include vital signs, completion of a symptoms 
review questionnaire, medical history, physical examination, anthropometric 
measurements (weight, height), screening clinical blood tests (HIV, HBV and HCV, 
complete metabolic panel, including glucose, fasting insulin to calculate HOMA-IR, liver 
function tests, complete blood counts, PT, PTT, and INR, fasting lipid panel, uric acid, 
HbA1c, and TSH (to assess thyroid function), screening urine drug test, urine pregnancy 
test (only in women who are able to become pregnant), and administration of the 
MMSE. The visit will last approximately 3 hours. About 28 mL of blood will be taken 
during the screening visit. If inclusion criteria are met, participants will participate in a 
Baseline Visit.  
 
At the end of the Screening visit, eligible participants will be given an accelerometer to 

 
 
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wear for 96 hours. They will be given instructions on how to wear the device and to mail 
it back to the unit via a pre-paid envelope. To ensure more accurate estimates of 
physical activity, participants will keep an activity log of when they sleep, exercise, or 
bathe. 
 
4.d. Study procedures 
 
4.d.1.Chronological ordering of procedures (see Table 1 and 2) 
 
All procedures are conducted solely for research purposes. This protocol has no 
relationship to other protocols. Radiation will be used only for fluoroscopy-guided 
Lumbar Puncture (LP) (up to twice for the duration of the study). To facilitate scheduling 
(e.g., if fluoroscopic LP cannot be scheduled on the same day) or satisfy a given 
participant’s request, we may split the various procedures of the Baseline Visit over two 
days within a week from each other. 
 
The study is comprised of: an initial telephone pre-screening to describe the study and 
inclusion/exclusion criteria to potential participants; a “Screening Visit”; randomization 
into one of two groups; a “Baseline Visit”; the “Outcome Visit” after 8 weeks. During the 
8-week period for the 5-2 CR or control groups, participants will return every two weeks 
for “Compliance Visits”, to assess and reinforce dietary compliance in keeping with 
previous protocols.39,40,94 The Outcome Visit will take place at the conclusion of the 8-
week phase. Specifically for the 5-2 CR group, the Outcome Visit will be timed during the 
second of the two CR days. In total, there will be 6 outpatient visits to the NIA Clinical 
Unit: Screening Visit, Baseline Visit, Week 2 Compliance Visit, Week 4 Compliance Visit, 
Week 6 Compliance Visit, and the Week 8 Outcome Visit. There will be an additional 
follow-up phone call or electronic mail within 3 days of completing the Week 8 Visit to 
assess any problems related to their final visit. Accelerometry will be implemented on 
two occasions for 96 hours each time: once between the screening and baseline visits 
and again after the Week 8 Outcome Visit. Throughout the study, additional safety visits 
may take place for any clinically significant. Participants will be involved in the study for 
approximately 8 – 11 weeks (allowing for an up to 3 week gap between Screening and 
Baseline Visits). Study participation will end with completion of accelerometry after the 
Week 8 Outcome Visit. 
 
It is anticipated that participants in this study will occasionally miss or fail to complete 
an assessment or procedure, such as a completion of a rating scale or a blood draw. 
Omissions such as these will be considered expected events and not protocol deviations 
provided they are infrequent and do not include data needed to assess safety or the 
primary study outcome. Cumulative proportions of these missed events in the study 
population will be presented to the IRB annually. In addition, the rate of omissions will 
be monitored by the Investigators. If an individual misses more than 15% of the required 
assessments/procedures or if more than 15% of the participants miss completion of the 
same assessment or procedure, it will be considered a deviation and a deviation report 

 
 
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will be sent to the IRB within two weeks.  
 
Table 1: Procedures 
  
Visit 1 
Visit 2    
Visit 3, 4, 5  
Visit 6    
  
Week 0 
Weeks 2, 4, 6  
Week 8 
 1   Day       
3-hours 
Day 1       
7-9 hours 
Day 2 
optional 
 1 Day            
1-hour 
Day 1       
7-9 hours 
Day 2 
optional  
Procedures 
Screening 
Baseline 
Compliance 
(Outpatient) 
Outcome 
Informed Consent 
X 
  
  
  
  
  
History and Physical (MMSE Visit 1 
only) 
X 
  
  
  
X 
  
Review Inclusion/Exclusion Criteria 
X 
X 
  
  
  
  
Symptoms Review Questionnaire 
X 
X 
  
  
X 
  
PANAS, RAND SF-36  
  
X 
  
X 
X 
  
Cognitive Testing 
  
X 
  
  
X 
  
MRI 
  
X 
  
  
X 
  
Accelerometers 
X 
  
  
  
X 
  
Vital Signs 
X 
X 
X 
X 
Anthropometric measurements 
X 
X 
  
X 
X 
  
Urine Drug Screen 
X 
  
  
  
  
  
Urine Pregnancy test 
X 
X 
 
 
X 
 
Fasting Blood Draw 
X 
X 
  
X 
X 
  
Mixed Meal Tolerance Test 
  
X 
  
  
X 
  
Lumbar Puncture 
  
X 
  
X 
Dispense Diet Shakes (5-2 CR Group) 
  
X 
  
X 
  
  
Dietary Counseling-ALL Groups 
  
X 
  
X 
  
  
Meals / Snacks 
  
X 
X 
X 
Follow-up Phone Call or Email 
  
Weeks 1, 3, 5, 
7 
Follow-up 
Payment 
$50.00  
$350.00  
$150.00* 
$500.00  
 
 
 
 

 
 
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Table 2: Laboratory 
  
Visit 1 
Visit 2    
Visit 3, 4, 5  
Visit 6    
  
Week 0 
Weeks 2, 4, 6  
Week 8 
 1   Day       
3-hours 
Day 1       
7-9 hours 
Day 2 
optional 
 1 Day           
1-hour 
Day 1       
7-9 hours 
Day 2 
optional  
Laboratory 
Screening 
Baseline                 
within 4 weeks of 
Screening Visit 
Compliance 
(Outpatient) 
Outcome 
Clinical Labs: CBC with differential, 
CMP, HbA1c, Phosphate, Magnesium, 
TSH, Liver Panel, Lipid Panel, Uric Acid, 
PT/INR, PTT, insulin, HIV, HBV, HCV 
X 
  
  
  
  
  
Research Lab: Insulin 
X 
 
 
 
 
 
Clinical Labs: CBC and PT/INR, PTT 
  
X 
  
  
  
  
Research Labs: AD Biomarkers, 
Cytokines/Chemokines, Neurotrophic 
Factors, Exosomes, Oxidative Stress 
Biomarkers, gene expression and DNA 
methylation, WBC differential counts, 
β-hydroxybuterate 
  
X 
  
  
  
  
CSF 
 
X 
 
X 
Research Labs: β-hydroxybuterate, 
gene expression and DNA methylation, 
WBC differential counts, plasma sample 
for AD Biomarkers, 
Cytokines/Chemokines, Neurotrophic 
Factors, Exosomes, Oxidative Stress 
Biomarkers 
 
 
 
X 
 
 
Clinical Labs: CBC with differential, 
CMP, HbA1c, Phosphate, Magnesium, 
TSH, Liver Panel, Lipid Panel, Uric Acid, 
PT/INR, PTT, insulin 
  
  
  
  
X 
  
Research Labs: AD Biomarkers, 
Cytokines/Chemokines, Neurotrophic 
Factors, Exosomes, Oxidative Stress 
Biomarkers, gene expression and DNA 
methylation, WBC differential counts, 
insulin, β-hydroxybuterate 
  
  
  
  
X 
  

 
 
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Amount of Blood 
28 ml 
 168 ml 
62 ml/visit 
174 ml 
 
4.d.2. Baseline visit  
 
This visit will occur within four weeks from the Screening Visit. Participants will arrive on 
the NIA-Clinical Research Unit after a 12-hour fast. The following procedures will be 
done: vital signs, anthropometric measurements (weight, waist circumference, sagittal 
diameter), and a blood draw for clinical labs (CBC, PT, PTT, and INR to ensure safety of 
the LP), urine pregnancy test (only in women who are able to become pregnant), and 
research labs including DNA collection (for APOE genotyping and gene expression 
studies). A 4-hour mixed meal tolerance test will be done. They will also have cognitive 
testing and will be asked to complete the Symptoms Review Questionnaire, PANAS, and 
RAND SF-36. A lumbar puncture (LP) will take place; up to a total of 15 mL of CSF will be 
collected. Participants will have a brain MRI. About 168 mL of blood will be taken during 
the visit. To facilitate scheduling or satisfy a given participant’s request, we may split the 
various procedures of the Baseline Visit over two days within a week from each other.  
 
4.d.3. 5-2 CR or control groups 
 
At the end of the Baseline Visit, all participants will receive informational materials and 
one-on-one dietary counseling about “healthy living”, consisting of providing and 
reviewing published material (by the CDC and USDA) on meal portion control, calories in 
common beverages, meal substitutions for controlling calorie intake, and other dietary 
considerations. Participants will then be randomized by study investigators into one of 
two groups to either 1) implement 8 weeks of 5-2 CR (cases); or 2) implement “healthy 
living” diet (controls).  
 
Participants in the 5-2 group will receive all shakes (Boost®, CWI Medical) from the NIA-
Clinical Research Unit.  
 
4.d.4. Compliance visits 
 
To assess and reinforce compliance with their respective diet, we will contact 
participants by phone or email (via the Secure Email & File Transfer Service: 
https://secureemail.nih.gov/bds/Login.do ) at weeks 1, 3, 5, 7 and four days after the 
Outcome Visit. In addition, we will conduct 2, 4, and 6 week Compliance Visits. The 
same procedure described below will occur during each one of these. 
 
These visits will take place during the second of the two consecutive restriction days for 
the 5-2 CR group or any day for the “healthy living” diet group after an 8 hour fast. A 
blood draw to measure β-hydroxybutyrate, vital signs and anthropometric 

 
 
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measurements (weight, waist circumference, sagittal diameter) will be done. 
Participants will provide feedback regarding physical and psychological symptoms by 
completing a Symptoms Review Questionnaire and the RAND SF-36. Samples for β-
hydroxybutyrate levels will be sent to Harbor Hospital Lab. In addition, a plasma sample 
will be sent to the NIA Core Lab for future studies. These visits will take about 1 hour.  
 
In addition, blood will be collected for isolation of total RNA and genomic DNA for target 
pathways gene expression and methylation analyses, and PBMCs counts. 62 ml of blood 
will be collected during each one of these visits. 
 
4.d.5.Outcome visit 
 
The Outcome Visit will occur at the end of the 8-week period in the 5-2 CR or control 
group. This visit will take place during the first of the two consecutive constriction days 
for 5-2 CR participants or at a convenient time during the week for control participants. 
This visit will occur after a 12-hour fast, to ensure comparability of the mixed meal test 
with the Baseline Visit. The visit procedures will be similar to the Baseline Visit, including 
a lumbar puncture (LP) and a medical history and physical exam.  
 
Participants will arrive on the NIA-Clinical Research Unit after a 12-hour fast. The 
following procedures will be done: vital signs, anthropometric measurements (weight, 
waist circumference, sagittal diameter), and a blood draw for clinical labs (CBC, PT, PTT, 
INR, CMP, lipid panel, uric acid, HgbA1c, insulin, β-hydroxybutyrate), urine pregnancy 
test (only in women who are able to become pregnant), and research labs including DNA 
collection (for gene expression studies). A 4-hour mixed meal tolerance test will be 
done. They will also have cognitive testing and will be asked to complete the Symptoms 
Review Questionnaire, PANAS, and RAND SF-36. A lumbar puncture (LP) will take place 
and up to a total of 15 mL of CSF will be collected. Participants will also have a brain 
MRI. About 174 mL of blood will be taken during each visit (including clinical and 
research labs). To facilitate scheduling or satisfy a given participant’s request, we may 
split the various procedures of the Outcome Visit over two days within a week from 
each other.  
 
At the end of the Outcome visit, participants will be given an accelerometer to wear for 
96 hours. They will be given instructions on how to wear the device and to mail it back 
to the unit via a pre-paid envelope. To ensure more accurate estimates of physical 
activity, participants will keep an activity log of when they sleep, exercise, or bathe. 
 
4.d.6. Procedures 
 
Medical history: A NIA licensed practitioner (Physician or Nurse Practitioner) will obtain 
standardized medical history including past medical history, current review of systems, 
medications and supplements, as well as family history of Alzheimer’s disease and 
dementia. 

 
 
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Physical examination: Vital signs including body temperature, blood pressure, and heart 
rate will be obtained. A NIA licensed practitioner (Physician or Nurse Practitioner) will 
conduct screening head, eyes, ears, nose, and throat (HEENT), cardiovascular, 
respiratory, abdominal, neurological exams. 
 
Anthropometric measurements: Height, weight, waist circumference, and sagittal 
diameter will be obtained with the subject in a hospital gown without shoes. 
 
PANAS: Participants will report on Likert scales how they feel emotionally. The PANAS is 
a common index of emotional state.71 
 
Symptoms Review Questionnaire: Participants will report on Likert scales their mood, 
level of energy, difficulty concentrating, headaches, feeling cold, constipation, negative 
emotions (depression, anxiety, irritability), hunger and preoccupation with food during 
constriction and non-constriction days for the week prior to the visit.  
 
RAND SF-36 scale: This questionnaire gauges physical and psychosocial effects of the 
dietary interventions. It has been used as a quality of life assessment in our previous 5-2 
CR study.40 
 
Screening blood tests: After a 12-hour fast, 28 mL of blood will be drawn for the 
following blood tests: complete metabolic panel, liver function tests, complete blood 
counts, PT, PTT, INR, fasting lipid panel, Hba1c, TSH, insulin, HIV, HBV, and HCV. 
 
Screening urine toxicology screen: Urine will be collected during the screening visit for a 
standard toxicology screen.  
 
Urine pregnancy test: A urine pregnancy test (only in women who are able to become 
pregnant) will be performed at screening and before MRI or fluoroscopic LP procedures 
at the Baseline and Outcome Visits. 
 
4-hour mixed meal tolerance test: After fasting for at least 12 hours, participants will be 
asked to drink 5 mL/Kg of the dietary supplement shake Ensure Plus®. Ensure Plus® is a 
mixed liquid meal; one bottle (480 mL) contains 95 g carbohydrate as dextrose, 26 g 
protein, and 25 g fat, providing 710 kilocalories. In addition, to inducing a change in 
glucose and insulin, the mixed meal tolerance test is ideal for testing changes in GLP-1, 
GIP, ghrelin, glucagon, pancreatic polypeptide, C-peptide, and other hormones. A blood 
draw will take place before subjects consuming the liquid meal Plasma, serum and 
whole blood biomarkers will be measured from this blood draw. for biomarkers, 
hormones, and cytokines (approximately 35 mL). Then subjects will ingest the liquid 
meal, followed by blood draws every 20 minutes (5 mL per sample). Typically, 
participants will have an intravenous (IV) line placed in an arm vein for blood draws.  

 
 
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Lumbar puncture (LP): Participants will have LPs performed during the Baseline Visit and 
Outcome Visit at 8 weeks, to provide CSF for measurement of biomarkers. Up to a total 
of 15 mL of CSF will be collected on each of those two visits. CBC, PT, and PTT results 
should be available prior to each LP taking place. Participants will be encouraged to be 
well hydrated on the day of the LP.  
In individuals in whom a landmark-guided LP at the bedside is deemed technically 
difficult by the medical staff (such as in the presence of degenerative lumbar spine 
disease, prior lumbar spine surgery, kyphosis/scoliosis or other conditions) or fails, a 
licensed Interventional Radiologist may perform the procedure under ultrasound or X-
ray guidance (fluoroscopy-guided LP).  
Cognitive testing: Logical Memory and CVLT will be administered using standard 
procedures. The MMSE will be administered only during the Screening Visit. In addition, 
we will administer EXAMINER, a software program 
(http://examiner.ucsf.edu/index.htm) implementing a battery of neuropsychological 
tests that reliably and validly assesses domains of executive function. This software was 
kindly given via a technology transfer request from Dr. Joel Kramer at the University of 
California – San Francisco. We will assess working memory, inhibition, set shifting, 
fluency, planning, insight, and social cognition and behavior. Neuropsychological testing 
will take approximately 1 hour to complete. 
 
Brain MRI: In total, structural MRI, fMRI and MRS will last about 2 hours. 
 
• Structural MRI -- Standard structural brain images will be collected for 
coregistration with fMRI and to allow for clinical over-read. 
 
• Functional MRI (fMRI) -- 
 
I. fMRI Study 1 will implement an event-related design, with two intermixed 
conditions (appetitive “junk” foods vs. non-appetitive foods), in two runs.  
Appetitive food trials will be pseudo-randomly intermixed with non-
appetitive food trials. Each trial will last for 4 seconds and will be followed by 
a variable interstimulus interval during which a fixation cross will be 
displayed. In each trial, they will be presented with a picture of a food item. 
Participants will be asked “how much they would like to eat” a given food, as 
it appears on screen by selecting one of 4 responses: ‘‘Really like it’’, ‘‘Like 
it,’’ ‘‘Do not like it,’’ or ‘‘Really do not like it.’’  Each of the 60 appetitive or 
non-appetitive food items will be shown about twice during each run.  
II. fMRI Study 2 will be a block design of a color-word Stroop task adapted from 
Nestor and colleagues.61 The study consists of congruent, incongruent, and 
rest blocks, presented over two counterbalanced runs. Each run consists of 8 
congruent blocks, 8 incongruent blocks, and 15 rest blocks. Each block 
consists of 15 trials. The order of blocks will cycle through a congruent block 

 
 
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to a rest block to an incongruent block; the pattern will then repeat. The 
counterbalanced run will begin in the opposite order and similarly cycle. 
During congruent or incongruent trials, the participant will be asked to select 
what the color of the word is on the screen: red, green, blue, or yellow.  
III. fMRI Study 3 will be a resting state fMRI study in a single run. Subjects will be 
asked to keep their eyes open and fixate on a cross. This study will be used to 
assess intrinsic functional connectivity between different brain regions.  
 
• MRS -- We will assess the concentrations of several brain metabolites, including 
glutamate, GABA, NAA, creatine, lactate, and glucose, using J-PRESS MRS, 
followed by fitting of metabolites using ProFit.63,95,96 Each of these metabolites 
has a characteristic pattern of spectral lines defined by the energy of transitions 
that occur when nuclei are excited into a higher energy state through radio 
frequency pulses. The spectral lines from all metabolites present, taken together, 
make up the observed spectrum. Concentrations of individual metabolites can 
be determined through evaluation of the relative sizes of the spectral lines in the 
observed spectra. The reliability of the measurement and fitting procedure for 
each metabolite can be assessed using Cramer-Rao lower bounds.97 We will use 
standard localization techniques based on magnetic field gradient sequences to 
obtain spectra from ventromedial prefrontal cortex and hippocampus.  
 
Accelerometry: Physical activity will be monitored using the Actigraph GT3X-BT 
(Actigraph Corporation) accelerometer. The Actigraph is a 1 x 1 x 3/8 inch device worn 
as a wristband on the non-dominate hand. The Actigraph GT3X-BT measures 
accelerations in vertical, horizontal, and anterior-posterior directions. We will sample at 
30 Hz per second over period of 96 hours at a time on two occasions during the study.  
Participants will be given a diary to record times for bed and times waking up daily, and 
time that the accelerometer was removed daily for extended periods of time. 
 
Gene expression array: Peripheral blood cells will be collected using the PAXgene 
system,98 to preserve transcript expression levels as they would be in vivo. Total RNA 
will be extracted using the PAXgene Blood mRNA kit (Qiagen, Crawley, UK) according to 
the manufacturer’s instructions. Whole genome expression profiling of the samples will 
be conducted using the Illumina Human HT-12 microarray (Illumina, San Diego, USA) as 
previously described.99 
 
Methylation arrays: One microgram of genomic DNA will be extracted from buffy coat 
samples was bisulfite converted using Zymo EZ-96 DNA Methylation Kit (Zymo Research 
Corp., Irvine, CA) per the manufacturer's protocol. CpG DNAm at individual sites will be 
assayed using the Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc., San 
Diego, CA) per the manufacturer's protocol. 
 
White blood cell differential counts: The RNA and DNA used for the gene expression and 
methylation assay will be isolated from mixed population cells. The standard complete 

 
 
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blood count provides percentages of neutrophils, lymphocytes, monocytes, eosinophils 
and basophils. We will require further separation of lymphocytes to get the counts of T 
cells, B cells and natural killer cells. The distribution of white blood cells will be an 
important covariate in the analysis of gene expression and methylation to ensure that 
the associations that we observe is specific to 5-2 CR rather than changes in any of the 
cells during the dietary intervention. We will use cell specific monoclonal antibodies and 
flow cytometry to count the each cell population within the lymphocytes. The flow 
cytometry methods will require 5-10cc of whole blood. 
 
4.e. End of participation 
 
We will share with participants any new information that may relate to their willingness 
to continue to participate in this study. Within 3 days after the Week 8 Outcome Visit, 
either one of the investigators or nursing staff will contact the participant to assess 
whether there have been any problems since the visit. In addition, at the end of the 
Outcome visit, participants will be given an accelerometer to wear for 96 hours. They 
will be given instructions on how to wear the device and to mail it back to the unit via a 
pre-paid envelope. Completion of accelerometry and the final telephone or email 
communication mark the end of participation to the study. 
 
Transfer of care or medical care offered at study completion is not applicable to this 
protocol, as the participants remain under the care of their primary health care 
providers.  
 
We will discuss the results of physical examination, clinical lab tests, and structural MRI 
with participants and provide them relevant reports. We will encourage subjects to 
share any clinically significant information with their health care providers.  
 
5. Management of Data and Samples  
 
5.a. Storage 
 
Samples and data collected under this protocol may be used to study physiology related 
to brain function, endocrinology and metabolism. The subject’s stored samples will be 
labeled with a code (such as letters and numbers) that only the study team can link to 
the subject. Any identifying information about the subject will be kept confidential to 
the extent permitted by law. Samples acquired during this study will be tracked using 
the NIA Biological Sample Inventory system following NIH guidelines.   
 
Samples of the subject’s blood and cerebrospinal fluid will be kept in the National 
Institutes on Aging, NIH Core Lab or one of our contract facilities. The subject’s samples 
may be tested immediately, or they may be frozen and used later. The subject’s samples 
will be stored with a confidential code. Samples may be kept until no cells remain or 
until the investigators decide to destroy them. If the subject gives us permission some 

 
 
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samples may be released to other doctors and scientists who are not associated with 
this institute. The Principal Investigator on this protocol will decide which co-
investigators and collaborating researchers may receive samples. The subject’s samples 
may be used in their research only if the research has been approved by an Institutional 
Review Board (IRB) and is related to the original research questions association with this 
protocol or for other research purposes as indicated below. 
 
We will code the blood, cerebrospinal fluid and urine samples that will keep the 
subject’s identity confidential. We will retain a code list that enables us to link the 
clinical information and results on each sample to the subject. DNA extraction from 
PBMCs will take place for APOE genotyping (only at Baseline Visit) and methylation 
studies and RNA extraction will take place for gene expression studies. DNA samples will 
be stored at the NIA Core Lab until they are released to the appropriate NIA lab. 
 
Access to research will be limited using either a locked room or a locked freezer. 
Samples and data will be stored using codes assigned by the investigators or their 
assigned designee(s). Data will be kept password-protected computers. Only 
investigators or their designee(s) will have access to the samples and data. 
 
At the completion of the protocol (termination), samples and data will either be 
destroyed, or after IRB approval, transferred to another existing protocol or a repository 
protocol. 
 
5.b.  Data  
 
Data and samples may be shared with collaborating laboratories at NIH or outside of 
NIH if consent for sharing was obtained in the original consent form. Samples/data that 
we may share in the future include biological specimens (blood, CSF), MRI images, MRS 
data, cognitive test results, and genomic data.  
 
Samples and data will be stripped of identifiers and may be coded (“de-identified”) or 
unlinked from an identifying code (“anonymized”). When coded data is shared, the key 
to the code will not be provided to collaborators, but will remain at NIH. Data and 
samples may be shared with investigators and institutions with an FWA or operating 
under the Declaration of Helsinki (DoH) and reported at the time of continuing review. 
Sharing with investigators without an FWA or not operating under the DoH will be 
submitted for prospective IRB approval. Submissions to NIH-sponsored or supported 
databases and repositories will be reported at the time of Continuing Review. 
Submission to non-NIH sponsored or supported databases and repositories will be 
submitted for prospective IRB approval. 
 
Required approvals from the collaborating institution will be obtained and materials will 
be shipped in accordance with NIH and federal regulations.  
 

 
 
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Whenever appropriate, the subjects will be provided with additional pertinent 
information after participation. For unlinked data, the identities of the participants will 
not be traceable and no later information can be provided. For coded samples, 
participants will be provided with additional pertinent information after participation 
whenever possible. Data and samples may also be shared with collaborating 
laboratories at NIH or outside of NIH and/or submitted to NIH-designated repositories 
and databases if consent for sharing was obtained in the original consent form.   
 
6. Additional Considerations 
 
6.a. Research with investigational drugs or devices 
 
This protocol does not include investigational drugs or devices. 
 
6.b. Gene Therapy 
 
Gene therapy is not used in this protocol. 
 
7. Risks and Discomforts 
 
 7.a. Medical history and physical exam: There are minimal risks to completing medical 
history and performing a physical exam. 
 
7.b. Anthropometric measurements: There are minimal risks in performing 
anthropometric measurements 
 
7.c. PANAS: There is minimal risk to completing the PANAS questionnaire 
 
7.d. Symptom Review Questionnaire: There is minimal risk to completing the symptom 
review questionnaire 
 
7.e. RAND SF-36: There is minimal risk to completing the RAND-SF 36 questionnaire 
 
7.f. Screening blood test: There is a slight risk of pain and bruising around the site where 
blood is drawn; bleeding and infection are rare side effects. To minimize this risk, the 
routine blood-drawing protocol will be followed and pressure will be applied to the site. 
Some people experience feelings of light-headedness or dizziness after having blood 
drawn. To reduce the risk of falling, we will monitor the subject closely and ask about 
these symptoms before we allow them to stand. The samples collected will be used to 
monitor the subject’s medical condition and for research purposes. 
 
7.g. Screening urine toxicology screen: A urine toxicology screen will be performed 
during the screening visit. If the drug test is positive the results of the drug testing will 
be noted in participants’ NIH medical record. Participants who do not want this 

 
 
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information in their medical records should not participate in the study. The medical 
records can only be released with written agreement by the participant. However, 
insurance companies may require individuals to release these records and may not 
provide insurance if they refuse.  
 
7.h. Urine pregnancy test: There is minimal risk to giving a urine specimen for pregnancy 
testing.  However, due to the unknown risk of MRI or fluoroscopic LP on pregnant 
women or fetal development, women who are able to become pregnant will have a 
pregnancy test prior to these procedures. Participants will not be able to participate in 
the study if the pregnancy test is positive.  
 
7.i. 4-hour mixed meal tolerance test: Drinking the Ensure Plus® shake may lead to a 
temporary sense of abdominal fullness and discomfort, nausea, vomiting, or diarrhea, 
but it is a rare occurrence. Needle insertion of the catheter will in rare cases produce 
blood at the site of insertion and bruising. 
 
7.j.Lumbar Puncture (LP): A lumbar puncture has a very small risk of local or CNS 
infection, local or epidural hematoma formation or CSF leak and hypotension. A 
minimum of about 5% of participants who have an LP may experience post-LP 
headache, which is often mild and responds to hydration and regular analgesics, but 
rarely may be severe and require hospitalization. In severe cases, such headache may 
require a blood-patch. 
 
To minimize the risk of infection, standard procedures for local anesthesia and 
antisepsis will be followed. To minimize the risk of post-LP headache, participants will be 
encouraged to present well-hydrated and only 15 mL of CSF will be collected. Moreover, 
an atraumatic needle will be used and the stylus will be reinserted prior to needle 
withdrawal as per current guidelines. Participants will be asked to remain in a reclining 
position for one hour and remain on the unit for three hours after the LP for 
observation. The LP may be aborted at any time at the participant’s request or after a 
limited number of unsuccessful attempts at the clinical judgment of the investigator 
performing the procedure. 
 
7.k. Radiation risk from fluoroscopy-guided lumbar puncture: We classify fluoroscopy-
guided LP as greater than minimal risk. This research study involves exposure to 
radiation from the above-mentioned procedure, which may be conducted up to two 
times (during Baseline and Outcome Visits), depending on whether a landmark-guided 
LP at the bedside is deemed technically difficult by the medical staff or fails. In addition, 
an ultrasound-guided LP may be attempted prior to the fluoroscopy-guided LP, at the 
discretion of the interventional radiologist who will perform the procedure. This 
radiation exposure is not required for medical care and is for research purposes only. 
The effective dose of radiation the participant will receive in this study is 0.74 rem, 
which is below the guidelines of 5 rem per year allowed for research subjects by the NIH 
radiation Safety Committee. The average person in the United States receives a 

 
 
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radiation exposure of 0.3 rem per year from natural sources, such as the sun, outer 
space, and the earth’s air and soil. While there is no direct evidence that the amount of 
exposure received from participating in this study is harmful, there is indirect evidence it 
may not be completely safe. There may be a very slight increase in the risk of cancer.  
 
The participant will be screened by the clinical research coordinator or study physician 
or NP for radiation exposure in the past year, either from other research studies or from 
medical test or care, to ensure they are within the guidelines. This will be done using a 
radiation exposure checklist screening form.  
 
7.l. Brain MRI: The Food and Drug Administration (FDA) has recently established safety 
criteria for human exposure to MRI studies. The MRI scanner used in this study satisfies 
these safety criteria. People are at risk for injury from the MRI magnet if they have 
pacemakers or other implanted electrical devices, brain stimulators, dental implants, 
aneurysm clips (metal clips on the wall of a large artery), metallic prostheses (including 
metal pins and rods, heart valves, and cochlear implants), permanent eyeliner, 
implanted delivery pump, or shrapnel fragments. Welders and metal workers are also at 
risk for injury because of possible small metal fragments in the eye of which they may 
be unaware. Participants will be asked to complete an MRI screening form for each MRI. 
If any participant is found not to be eligible for the MRI or refuse the MRI, they will be 
disqualified from the study. In addition, all magnetic objects (for example, watches, 
coins, jewelry, and credit cards) will be removed before entering the MRI scan room. 
Participants with history of surgeries, implanted devices, etc. will be asked to provide us 
with surgical reports mentioning the type of the clips, prostheses, etc. used. Every effort 
will be made to establish the MR compatibility or incompatibility of these implants, 
according to the safety standards of the NIA 3T MRI center, including obtaining reports 
by manufacturing companies for MR compatibility. The research staff will evaluate each 
person with a less severe history of claustrophobia on a case-by-case basis. 
 
Regarding minor discomforts, radiofrequency pulses emitted by the MRI machine can 
induce mild muscle twitching in some participants. If the sensation becomes overly 
uncomfortable, the scan will be stopped. The loud sounds (> 120 dB) emitted by the MRI 
machine during scanning require earplugs but may cause mild acoustic discomfort.  
 
7.m. Cognitive Testing: Occasionally, cognitive tests may be tiring or stressful. 
Psychological testing may cause some people to feel anxious. 
 
7.n. Genetic testing (APOE genotyping and DNA methylation studies): The DNA analysis 
that will be done as part of this study is done for research purposes only.  
 
Genetic testing can provide information about heritability of diseases and risk factors. 
Knowledge that participants or their children are at increased risk of disease may cause 
anxiety or depression or make them reevaluate their life. This is the case of the APOE ε4 
gene, for which we test in this study and which increases the risk of Alzheimer’s disease 

 
 
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and cardiovascular disease, although it does not mean that whomever carries that gene 
will develop Alzheimer’s disease or coronary artery disease.  
 
As a result of genetic testing performed on a participant, her/his biological relatives may 
find out that they are at risk for a disease, such as Alzheimer’s disease, which might 
affect the participant’s relationships with them, cause stress, anxiety, or depression.   
 
Genetic counseling is available at the National Institutes of Health to help participants 
understand the nature and implications of genetic findings for them and their families. 
 
Because of the emotional risk, some people who participate in research do not want to 
know the results of genetic testing. It is our policy to not disclose the results of genetic 
testing unless it may have direct medical or reproductive implications for participants or 
their families. Participants may choose to receive their information or they may choose 
not to receive the information. Whether they choose to receive the information or not, 
by agreeing to participate in this study, participants do not waive any rights that they 
may have regarding access to and disclosure of your records.  
 
Results of genetic testing obtained at NIH are often preliminary and difficult to interpret 
because the testing is being done for research purposes only and the laboratories are 
not clinically certified. This is the case of the testing for the APOE ε4 gene that will take 
place in this study. In addition, testing for the APOE ε4 gene will take place after the end 
of the participants’ study participation and will not be immediately available for review. 
If participants decide that they wish to be informed about the results of APOE ε4 genetic 
testing as conducted in this study and this result has become available, they will meet 
with the principal investigator, who will refer them to a CLIA-certified lab for 
confirmation. In addition, the principal investigator will discuss the significance of their 
particular genotype on AD and cardiovascular risk for participants and their families 
based on the literature.   
 
Participants’ genetic information will be kept confidential to the extent possible. The 
results of participants’ genetic testing will be kept in a locked and secured manner at the 
NIH. Genetic information will not be part of participants’ medical record. Genetic 
information about participants will not be revealed to others, including their relatives, 
without participants’ written permission. Similarly, participants will not receive 
information about other family members.  
 
Problems, such as with insurance or employment discrimination, may occur if 
participants disclose information about themselves or agree to have their research 
records released.  We will not release any information about participants or their 
families to any physician, insurance company or employer unless you sign a document 
allowing release of the information. 
 
Further discussion can be found under Section “20. Confidentiality” on pages 51-52. 

 
 
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7.o. Accelerometry: The accelerometer has no risks associated with its use and causes 
minimal inconvenience. 
 
7.p. 5-2 CR/Control Diet: Overall, a minority of participants (9-15%) is expected to 
develop minor physical and psychological AEs during the first several weeks of 5-2 CR.  
Such AEs include lethargy, feeling cold, difficulty concentrating, and irritability. 
Participants may experience minor psychological symptoms such as feeling hungry 
(leading to preoccupation with food), lack of concentration, bad temper and irritability. 
The Harvie et al. study is especially important because our study design is similar to it.  
In the Harvie et al. study, a small number of participants on the 5-2 CR regimen (8%) 
reported physical symptoms including lack of energy, headaches, feeling cold, and 
constipation. Minor psychological symptoms included lack of concentration, bad 
temper, and a preoccupation with food among 15% of the 5-2 CR group. These adverse 
effects abated after cessation of CR.  Conversely, 6% of 5-2 CR participants reported 
better health and increased energy.40 Furthermore, 32% of 5-2 CR had increased self-
confidence and positive mood. To minimize these risks, participants will be told ahead 
of time about potential side effects. During Compliance Visits, we will give participants 
the opportunity to discuss and will offer encouragement that may ameliorate minor 
psychological symptoms.   
 
No major adverse effects have been associated with mild to moderate intermittent 
calorie restriction.39,40,94 Two of our previous CR studies did not have a reported serious 
AE incident.39,40 More severe caloric restriction in humans relative to our studies also 
produced no serious AEs 94. We classify the 5-2 CR and control diet as minimal risk. 
 
8. Subject safety monitoring 
 
8.a. Monitoring of Subjects 
The investigator or clinical staff performing a study procedure will monitor subject safety. 
This includes physicians, licensed practitioners (NPs) and RNs during LPs, blood draws, mixed 
meal testing, and History and Physical sessions; investigators administering questionnaires 
and cognitive testing, MRI techs and investigators during MRIs.  
 
The PI will make determinations regarding the presence and grading of on adverse events 
(AEs) and unanticipated problems (UPs). Whenever study investigators contact subjects 
throughout the study, they will seek information on AEs and UPs. AE’s and UPs will also 
be elicited during study visits, by subjects filling in the symptoms review questionnaire 
or during history and physical examination. Participants will be asked to use electronic mail 
(Secure Email & File Transfer Service: https://secureemail.nih.gov/bds/Login.do) or 
telephone to inform the investigators about any AE’s or UPs experienced. AEs and UPs may 
be observed by the Investigator and/or study staff, elicited from the subject and/or 
family member, or volunteered by the study subject. AEs and UP’s that had previously 
been reported by study subject will also be reassessed for duration, intensity and 

 
 
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possible reoccurrence. Assessment of safety will include vital signs, history and physical 
sessions, questionnaires on physical and psychological symptoms and clinical labs as 
indicated. Any AE or UP that occurs between the times a study participant signs the 
informed consent form and the end of participation will be assessed and recorded under 
NIH HRPP SOP # 16 and 16 A.  
 
In particular, we will monitor the weight loss of the participant. If a participant on 5-2 CR 
loses 4-5% of their body weight within a 2-week period between visits, additional safety 
visits will be scheduled every week to monitor body weight more closely. If weight loss 
in that range or greater continues to occur, participants will be removed from the study.  
 
A study participant will also be discontinued from the study if: 
 
• Any adverse event occurs Grade 2 or greater and possibly related to the study. 
• The participant develops any of the exclusion criteria. 
• There is persistent non-compliance with study requirements (keeping follow-up 
visits). 
Participants will be informed about new information from this or other studies that may 
affect their health, welfare, or willingness to stay in this study. Participants may decide to 
stop participating at any time. Any data or blood collected until that point in time will 
remain part of the study and are the property of the National Institute on Aging.  
 
9. Outcome measures 
 
All secondary outcome variables in this pilot study are exploratory. 
 
9. a. Primary outcome measures 
• Exosomal p-S312-IRS-1/p-panY-IRS-1 ratio 
 
9. b. Secondary outcome measures 
 
• Other exosomal protein and mRNA biomarkers of AD; 
• SI and HOMA-IR, indices of insulin resistance, derived from insulin and glucose 
values taken from a fasting blood draw; 
• fMRI activation to appetitive food versus non-appetitive food stimuli; 
• Resting state fMRI intrinsic functional connectivity of DMN; 
• Behavioral performance and fMRI activation during a Color-Word Stroop task; 
• Brain metabolites assessed with Magnetic Resonance spectroscopy and regional 
perfusion assessed with arterial spin labeling; 
• Memory and executive function scores. 
• Anthropometric measures (weight, waist circumference, sagittal diameter); 

 
 
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• Clinical Labs: lipid panel, uric acid;  
• Plasma ketones, such as β-hydroxybutyrate and other metabolites. 
• Plasma metabolites and hormones (GLP-1, insulin) during a mixed meal test; 
• CSF and plasma AD biomarkers, including Aβ1-42 and p181Tau;  
• Plasma and CSF neurotrophic factors, such as BDNF; 
• Plasma and CSF reactive oxygen species, advanced glycation end-products and 
advanced oxidation protein products; 
• Plasma and CSF pro-inflammatory cytokines, such as IL-6, IL-8, IL-12 and IL-23; 
• Ratings of symptoms that can occur with 5-2 CR, mood and quality of life 
changes, recorded during both the 5-2 CR and control diet phases; 
• Physical activity measured using an accelerometer. 
• Global gene expression pattern, mitochondria gene expression and expression of 
other gene pathways responding to CR 
• Genome-wide methylation in peripheral blood mononuclear cells  
 
10. Statistical Analysis 
 
10. a. Analysis of data/study outcomes 
 
This is a pilot study that is exploratory in nature and one of its goals is to collect 
preliminary data for multiple outcomes that may be used in future studies on the brain 
effects of CR.  
 
For the primary and each secondary scalar measure, a separate repeated measures 
mixed models analysis will be conducted to examine the effects of the between-subjects 
factor “Group” (5-2 CR group vs. “Healthy living” control group), the within-subjects 
factor “Time” (Baseline vs. Outcome) and their Interaction.  An Auto-Regressive (AR)1 
covariance matrix and Restricted Maximum Likelihood Estimation (REML) will be used. 
We will assess fit to the model using the AIC and BIC criteria (lower values indicate 
better fit). These parameters were chosen because they are recommended for repeated 
measures analyses, including fMRI task analyses.100 Regarding within-subjects effects, 
we will assess post-hoc differences between the 8-week outcome visit and baseline for 
the 5-2 CR and control groups and for each group separately (i.e. post-hoc tests for the 
Group * Time interaction). This analytic approach is common in the CR literature.101 
Mixed model post-hoc tests will employ the Least Significant Difference (LSD) statistic. 
We will use one-tailed t-tests. 
 
Criteria for significance will be set at alpha of 0.05 for all non-MRI analyses, and 0.005 
uncorrected at the voxel level for fMRI analyses. Given that this pilot study is 
exploratory, we will not conduct correction of multiple comparisons for the various 
secondary outcomes.  
 
Analysis of fMRI data will use a mixed model design to assess the effect of Group on 

 
 
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brain activation associated with viewing appetitive vs. non-appetitive food, the Stroop 
effect in the Stroop task, and DMN intrinsic functional connectivity. Analyses will be 
conducted at the whole brain level and within ROIs (vmPFC, OFC and insula for the fMRI 
food task, lateral and dmPFC for the Stroop task, and in DMN for resting state. The fMRI 
analyses will be corrected for type 1 error as the cluster level using Monte Carlo 
simulations.   
 
For brain MRS measures, a repeated measures analysis will be separately used for each 
of the brain regions of interest: the hippocampus and the prefrontal cortex.  
 
APOE genotype is a common covariate used when assessing populations with AD or at 
risk for AD.  We intend to perform exploratory analyses including APOE, which may 
reduce error variance and improve our statistical models.  
 
Plasma ketones are strongly affected by alternate day CR acutely but not chronically;39 
our main objective for measuring them is to assess compliance with 5-2 CR.  
 
We do not anticipate a change in physical activity in 5-2 CR versus “healthy living” 
control groups, because no change has been noted in our previous studies. Nonetheless, 
this is a common analysis requested by reviewers, to make sure that exercise effects did 
not contaminate diet effects.  
 
The data from the gene array and DNA methylation array will be analyzed using 
residuals after adjustment for white blood cell differential count in linear regression 
models. We will take two approaches to analyze the data. First we will focus on 
candidate genes approach where we select specific expression probe or CpG sites; DNA 
for these studies will be collected during Baseline, Compliance and Outcome Visits. 
Second, we will run genome-wide association analysis for differences in cross sectional 
and longitudinal changes in gene expression or DNA methylation between the control 
and 5-2 CR group at each gene array probe or CpG site. DNA for genome-wide 
association analyses will only be collected during Baseline and Outcome Visits. For the 
cross sectional analyses, we will use a paired t-test at each visit, and mixed models for 
longitudinal analysis. In addition, we will look at overall patterns of gene expression and 
methylation using principle components, DIANE and hierarchical clustering tools to 
determine whether there are specific gene expression and methylation signature in 
association with 5-2 CR. As secondary analyses, we will also determine whether gene 
expression and DNA methylation patterns can predict the changes in metabolic, 
cognitive and neurochemical variables collected in this study. Within the 5-2 CR group, 
we will test the association between baseline levels of gene expression or DNA 
methylations at each probe/CpG site with longitudinal changes in the main outcome 
variables using mixed models. 
 
10. b. Power analysis 
 

 
 
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The accrual number request is 100 participants. Our target sample size for completing 
the study is N = 40. There will be 20 participants in each group. We anticipate up to 60 
participants will not meet inclusion criteria during the screening visit or may withdraw 
from the study. Enrollment will continue and withdrawals will be replaced until we have 
acquired complete data from 40 participants.  
 
For power calculations, we used G*Power (http://www.gpower.hhu.de/en.html). 
 
First, we calculated power for the primary outcome (exosomal p-S312-IRS-1/p-panY-IRS-
1 ratio, which is a measure of brain IR). Effect size (f) was determined based on studies 
with comparable sample sizes examining moderate CR (20-40%) over a 2-3 month 
period in humans. 
 
Insulin Sensitivity (Peripheral and Brain): It is important to re-emphasize that reductions 
in fat mass will increase peripheral insulin sensitivity.12,21,22 To determine effect size, we 
examined a study by Weiss and colleagues101 that had a design similar to our own. They 
examined sedentary men and women aged 50-60 with a BMI of 23.5 to 29.9. They 
randomly assigned participants to moderate CR (n = 18) or a healthy lifestyle control 
group (n = 10) for 12 months. The CR group showed a significantly reduced BMI from 
27.1 to 24.8, with a corresponding increase in Si from 4.5 to 7.4. The healthy living 
control group showed a non-significantly reduced BMI from 27.9 to 27.4, with a non-
significant increase in Si from 4.2 to 4.5. The effect size was f = 0.440. Our most recent 
study on intermittent CR40 showed a reduction of body fat from 33.6kg to 30.6kg by 3 
mo., but a relatively smaller decrease (29.1kg) by 6 mo., suggesting that most of the 
weight loss occurred in the first three months. Based on these findings, we anticipate a 
comparable effect size (0.3) from our 8 weeks of 5-2 CR. 
 
Regarding our primary outcome (exosomal p-Ser312-IRS-1/p-panY-IRS-1), in our 
published study, we examined differences between preclinical samples from 
participants who eventually developed AD (92.1 ± 7.7) and controls (14.7 ± 0.9)51. We 
base our power analysis on the assumptions that the our subjects at high risk for AD had 
intermediate high values for p-Ser312-IRS-1/p-panY-IRS-1 ratio at baseline (ratio = 46), 
that the 5-2 CR intervention had a similar modest effect size as for peripheral IR in 
decreasing the ratio (0.3), whereas the control intervention induced no change. We 
used the following input parameters to determine power for ANOVA repeated 
measures, between-within interactions: α = .05 (one tailed); 2 groups (5-2 CR vs. Healthy 
Living diet); 2 repeated measures (Baselive vs. Outcome); correlation between repeated 
measures = 0.4. Based on these parameters, we achieve over 90% power with a total 40 
participants (see Figure 4 below for post-hoc power calculation (achieved power, given 
certain N and effect size)). These calculations are conservative (e.g. the effect size may 
be greater than 0.3); therefore, it is likely that we will actually achieve greater power in 
this study.  
 

 
 
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Figure 4. Post-hoc power calculation for primary outcome (exosomal p-Ser312-IRS-1/p-
panY-IRS-1) analyzed with ANOVA for repeated-measures for within-between 
interaction. The x-axis depicts total sample size and the y-axis depicts Power. 
 
In addition, we performed power calculations for several key secondary outcomes of 
interest.  
 
Regarding fMRI, we performed power calculations based on a series of equations by 
Mumford and Nichols100 that take into account the unique nature of fMRI data. We use 
an alpha = .005 for fMRI analyses, as recommended by Mumford and Nichols. 
 
fMRI task 1 (appetitive foods): A recent study by Bruce et al.102 examined young to 
middle-aged obese participants that either underwent 3 mo. of moderate CR (n=16) or 
bariatric surgery (n=15). They assessed fMRI activation to pictures of food versus 
pictures of animals at baseline and 3 mo. They found that CR participants showed a 
large cluster-wide increase in medial prefrontal cortex versus bariatric participants (t = 
3.72, Cohen’s d = 1.34 and f = 0.8), a brain region that they argue is involved in 
regulating appetite and suppressing cravings. To achieve at least 80% power with 20 
subjects per group in a between subjects contrast, we would require a scan time of 6 
min. for the fMRI task. Our scan time of 24 min. well exceeds this estimate and would 
achieve power of roughly 85-90%.  
 
fMRI task 3 (resting state fMRI): Zhang et al.103 recently published a proof-of-concept 
study on the acute effect of intranasal insulin (40 IU) on 14 subjects with type 2 diabetes 
and 14 healthy participants. Intranasal insulin, like CR, increases insulin availability in the 
brain.104 They in part examined changes in resting state fMRI brain activity during 
placebo (day 1) and intranasal insulin (day 2 or 3), collapsed across groups. They found 

 
 
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robust activation in several brain networks, including a portion of DMN in a medial PFC 
area (t = 3.25, Cohen’s d = 1.25 and f = 0.626) similar to what is described above for 
Bruce et al.102 To the best of our knowledge, this is the only published study examining 
the effect of a therapeutic intervention manipulating brain insulin on resting state 
activity. We anticipate a similar activation pattern and effect size in our data. According 
to calculations based on Mumford and Nichols100 for power estimation in fMRI, groups 
with n=20 each would require a 6-minute resting state scan to achieve 80% power. Our 
resting state scan lasts for approximately 7.5 minutes. 
 
Memory and Executive Function: Witte and colleagues65 examined aged participants 
divided into groups that either engaged in 3 months of moderate (30%) CR (n=20), 
received no intervention (n=10), or received unsaturated fatty acid supplementation 
(n=20). For the Rey Auditory Verbal Learning Test, which is very similar to our CVLT, CR 
participants showed a significant 130% gain (Cohen’s d = 0.799) in a composite memory 
score after the intervention versus non-significant changes in the other groups. Our own 
intervention only differs in the length of time spent on CR and the intermittent nature of 
the CR, and so we anticipate a comparable effect size. Given that executive function is 
more consistently impacted by obesity and by extension IR across the lifespan,105,106 we 
also anticipate a similar or greater effect size for executive function, although there are 
currently no studies in the literature directly comparable to our design. In summary, 
with an anticipated effect size f = 0.4, we will have power = 0.81 to detect an effect on 
cognition.  
 
Plasma biomarkers: For plasma biomarkers, such as proinflammatory cytokines, ROS, 
BDNF, and affect measures, we note that our previous within-subjects study on 
intermittent CR in humans39 produced robust differences in significant or near-
significant trends in these measures by 3 mo. with n = 42 at alpha = .05. We anticipate 
being able to detect such effects and more subtle differences, because our between-
subjects design is more powerful. In particular, informal power calculations suggest that 
we will have at least 80% power to detect differences in cytokines, ROS, and BDNF.  
 
Global gene expression and genome-wide methylation: With sample size of 20 for each 
of the two groups, using the Illumina HT12 expression chip with 25,000 annotated 
genes, there is 80% power at alpha (two-tailed) = 0.05 to detect a 1.5-fold difference in 
expression. Similarly, with the sample size of the proposed study, we have 90% power to 
detect methylation differences of 40% between groups at alpha (two-tailed) = 0.05 for 
45,000 CpG sites. 
 
11. Human subjects protection 
 
11. a. Subject selection 
 
Statement of equitability 
Participants will be recruited and enrolled equitably including, but not limited to, 

 
 
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without regard to race, ethnicity, sex, sexual orientation, unless explained below. 
 
It is National Institute on Aging (NIA) policy that the regulations of the Department of 
Health and Human Services (HHS), set forth in 45 CFR Part 46, are applicable to all 
research involving human subjects, as defined by these regulations, for which the NIA is 
responsible, regardless of the source of funding or whether the research is funded. In 
the case of conflict between regulations of the funding or regulatory agency and HHS, 
the more restrictive regulations shall prevail. The NIA is also obligated by law to adhere 
to the regulations of the Food and Drug Administration (21 CFR Parts 50 and 56) 
governing projects involving investigational new drugs [within the meaning of 21 U.S.C. 
sections 355(i) or 357(d)], or investigational new devices [within the meaning of 21 
U.S.C. section 360(g)]. 
 
Rationale for non-equitable selection 
 
This study imposes certain age and BMI limits to define a suitable population of older 
overweight adults with IR, which is suitable for addressing the study hypotheses, as 
explained in the introduction. The main reason we study participants aged 55-70 years 
who are overweight and have IR is to examine brain IR and other AD-associated 
biomarkers at individuals at higher risk for disease during the preclinical stage of AD. We 
impose a lower limit of BMI to ensure that we will recruit a population with high 
probability of IR. IR is present in many middle-aged23 and aged24,25 adults with a BMI 
above 27.26 Specifically, higher visceral adiposity due to being overweight or obese 
corresponds to higher IR in 25-40% of late middle-aged to geriatric Caucasian42 and 
Asian27 populations. Overweight to obese participants (BMI ≥ 27) aged 55-70 years have 
insulin sensitivity values of approximately 7.6 min−1/(μU mL−1) × 10−4.24,25 In addition, we 
impose the upper limit of 350 pounds for body weight, because this is the limit of the 
scanner. In addition, we exclude pregnant women, given the fact that we employ brain 
MRI and XRays for fluoroscopy-guided LPs, which may pose a higher risk to the unborn 
fetus. We exclude nursing women because they have higher caloric needs. 
 
11. b. Justification for exclusion of children 
 
The goal of this study is to provide proof of concept that 5-2 CR compared to dietary 
counseling can lower IR and ameliorate any detrimental effects on middle to older age 
cognitive performance, brain metabolism and function and normalize any biomarker 
abnormalities associated with higher risk of AD.  Children do not develop AD, and there 
are no data to suggest that IR during childhood may impact future AD risk. In addition, 
5-2 CR in juvenile animals permanently stunts their physical growth. Children are, 
therefore, not an appropriate population to examine for the purposes of our study. 
 
11. c. Justification for exclusion of other vulnerable participants 
 
If the present study provides proof of concept that 5-2 CR compared to dietary 

 
 
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counseling improves cognitive performance, brain metabolism and function and 
normalizes biomarker abnormalities associated with higher risk of AD, it would “pave 
the way” for testing it in cohorts with age-related cognitive impairment or AD. The study 
requires following strict instructions for 5-2 CR diet, which participants with cognitive 
impairment would find hard to follow. In addition, this is a proof of concept study that 
involves more than minimal risk without direct benefit. The study involves some minor 
but real discomfort (e.g. feeling hungry) and invasive procedures (such as LP), which are 
not appropriate for those without consent capacity in this study. 
 
11. d. Justification of sensitive procedures 
 
The experimental paradigm does not involve the use of deception, or other sensitive 
procedures.  
 
11. e. Safeguards for vulnerable populations 
 
We will perform pregnancy tests and exclude pregnant (and nursing) women. 
 
11. f. Qualifications of investigators 
 
The Principal Investigator has verified that all individuals working on this protocol 
required to take HRPP training under OHSRP SOP 25 (Training requirements for the NIH 
Human Research Protections Program) have completed all required training. 
 
Dimitrios Kapogiannis, M.D. (LCI/NIA) 
 
Dr. Kapogiannis is a Board-certified neurologist with subspecialty training on dementia 
and Experimental Therapeutics in Neurology. He will be principal investigator and 
medically responsible investigator of the protocol. He has been primarily responsible for 
the design of the study and will oversee the project, ensure quality assurance of the 
data and analyze the data. He will obtain consent, conduct History and Physicals and 
perform LPs. 
 
Mark Mattson, Ph.D. (LNS/NIA) 
 
Dr. Mattson is the Chief of the Laboratory of Neurosciences. He will be an associate 
investigator in this protocol. Dr. Mattson has conducted animal studies examining the 
neurobiology of CR. More recently, he has collaborated with several groups to look at 
the long-term effects of continuous and intermittent CR on CSF biomarkers, oxidative 
stress, psychological wellbeing, and other factors.39,40 He has also been primarily 
responsible for the design of the study. In addition, his lab will perform measurements 
of biomarkers in plasma and CSF and analyze data.  
 
Josephine Egan, M.D. (LCI/NIA) 

 
 
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Dr. Egan is the Clinical Director of the NIA and the Chief of the Laboratory of Clinical 
Investigation. Dr. Egan will be an associate investigator on this protocol.  Dr. Egan is an 
endocrinologist with extensive experience conducting and overseeing clinical research 
studies. She has contributed to the design of the study and will oversee its conduct to 
ensure participants’ safety. Her lab will perform measurements of biomarkers in plasma 
and CSF and analyze data. She will obtain consent, and conduct History and Physicals. 
 
Tamara Harris, M.D. (LEDB/NIA) 
 
Dr. Harris is the Chief of the Geriatric Epidemiology Section at the NIA. She has 
contributed to the design of the study. She will analyze accelerometry data. 
 
Toshiko Tanaka, Ph.D (TGB/NIA) 
 
Dr. Tanaka is associate investigator of the study and has experience performing genetic 
analyses in large populations. She will analyze DNA methylation and gene expression 
data. 
 
Luigi Ferrucci, M.D., Ph.D. (TGB/NIA) 
 
Dr. Ferrucci is the NIA Scientific Director. Dr. Ferrucci has lead efforts to investigate the 
gene expression and DNA methylation changes that occur with age in observational 
studies of aging. He will analyze DNA methylation and gene expression data. 
 
 
Onyinye Erondu, M.S., R.N. 
Mrs. Onyinye is the Clinical Research Coordinator for the study; she has more than 14 
years of clinical research experience. She will acquire consent, implement study 
procedures, including performing blood draws, mixed meal tolerance test, and assisting 
with the LP. 
 
Seema Gulyani, Ph.D., CRNP. (LNS, NIA/NIH) 
 
Dr. Gulyani is a Senior Research Fellow in the Laboratory of Neuroscience. She has 
completed her post-doctoral training in Neurosciences at University of California Los 
Angeles. She is also a licensed Nurse Practitioner and has practiced as a NP in the Johns 
Hopkins Neurology.  Dr. Gulyani will be lead investigator on the protocol. She will be 
responsible for multiple clinical duties along with PI including overseeing participants’ 
safety. She will obtain consent, conduct History and Physicals and perform LPs. 
 
Roger Mullins, MA, Ph.D. (LNS/NIA)  
 
Dr. Mullins is a Postdoctoral Fellow in the Human Neuroscience Unit under the training 
of Dr. Kapogiannis. He will be responsible for recruitment and contact with participants, 

 
 
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cognitive testing, MRI/fMRI/MRS, and data analysis. Dr. Mullins has over 5 years of 
experience with neuropsych assessment and MRI research on patients with 
neurodegenerative diseases 
 
12. Anticipated benefit 
 
This pilot study of two short-term diets is not expected to generate direct benefits for 
the participant. While the diets are likely to decrease body weight and increase insulin 
sensitivity (i.e., decrease HOMA-IR), changes which may be desirable to the participants 
and their physicians, the changes are likely to be temporary. This is a short-term study 
and, therefore, the magnitude and duration of these effects and their impact on long-
term health is unclear. The study will generate generalizable knowledge on the effects 
of short-term implementation of 5-2 CR. 
 
13. Classification of Risk 
 
13. a. For adults 
 
Overall, we classify the study as greater than minimal risk.  
 
13. b. For adults without consent capacity 
 
Not applicable. 
 
13. c. For children 
 
Not applicable. 
 
13. d. Overall risk and benefit consideration 
 
The risks are reasonable in relation to anticipated benefit.  
 
14. Consent Documents and Process 
 
14. a. Designation of those obtaining consent 
 
The principal investigator, clinical research coordinator (nurse) and NIA research nurses 
and nurse practitioners will be authorized to obtain consent. Investigators identified as 
being authorized to obtain consent in section 11.f. above will be allowed to obtain 
consent.  In addition, NIA research nurses and nurse practitioners are qualified to obtain 
consent by their training and will also be authorized to obtain consent. All staff 
obtaining informed consent has completed the NIMH HSPU “Elements of Successful 
Informed Consent” training. 
 

 
 
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14. b. Consent procedures 
 
All participants will receive a verbal explanation in terms suited to their comprehension 
of the purposes, procedures and potential risk of the study and of their rights as 
research participants.  Participants will have the opportunity to carefully review the 
written consent form and ask questions regarding this study prior to signing.    
When the participant fully understands her responsibilities and possible risks and 
discomforts and agrees to participate in the current study they will be provided with the 
consent to sign. A signed copy will be provided to each participant, as well as placed in 
her medical record. No procedures will begin until the informed consent has been 
properly obtained. 
 
In particular, the summary study design will be detailed. Furthermore, participants will 
be fully informed about the data and safety monitoring procedures.   
 
14. c. Consent documents 
 
Consent forms contain all required elements specific to the CNS IRB protocol template 
(rev 1.27.14). As stated, a consent document for healthy volunteers is submitted with 
this protocol. No special documents are needed. 
 
15. Data and Safety Monitoring 
 
15. a. Data and safety monitor 
 
The Principal Investigator (PI) will be the data and safety monitor.  
 
15. b. Data and safety monitoring plan 
 
The PI will conduct periodic review of the data every at least 3 months to ensure that 
data are collected, saved and stored in a consistent manner and according to the 
Protocol. The PI will conduct periodic review of safety data (clinically significant lab 
results, H&P sessions, procedure notes, etc.) every at least 3 months, including review of 
expected AE’s occurring more frequently than expected or at greater severity than 
expected.  
 
15. c. Criteria for stopping the study or suspending enrollment or       
procedures 
 
Temporary suspension of enrollment will occur if any related AE of grade 3 or higher 
occurs, in anticipation of its review by the NIA Clinical Director and the IRB. Study 
procedures for participants already enrolled may continue, unless otherwise instructed 
by the NIA Clinical Director and/or the IRB. The study may resume enrollment after the 
NIA Clinical Director and the IRB have given us permission to do so. 

 
 
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16. Quality assurance 
 
16. a. Quality assurance monitor 
 
The PI will be responsible for QA monitoring of this study. The PI will address any issues 
discovered. In addition the NIA Clinical Director may randomly review charts for Quality 
assurance. 
 
16. b. Quality assurance plan 
 
The PI will acquaint the research team with the basic principles of the ICH-GCP 
guidelines. The Pi will ensure that the research team is following the protocol, 
complying with regulatory policies, and collecting and reporting data according to the 
protocol. The PI will delegate to an experienced research professional to conduct audit 
on 10-20% of participant’s research charts once the participant has completed the 
study. In addition the Clinical Director may randomly review charts for quality 
assurance. The Quality Assurance designee will assess adherence to study procedures 
and timelines; review roles/responsibilities of research team members; clinical and 
research data collection; any AE or UPs. The PI will address any issues discovered.   
 
17. Reporting of Unanticipated Problems, Adverse Events and Protocol  
Deviations 
 
The Principal Investigator is responsible for detecting, documenting, and reporting 
unanticipated problems, adverse events (AEs), including serious adverse events (SAEs), 
and deviations in accordance with NIH policy, IRB requirements, and federal regulations. 
Relatedness to the research of all serious adverse events will be determined by the PI in 
consultation with the Clinical Director. 
 
Serious unanticipated problems, serious adverse events (including deaths) that are not 
unanticipated problems, and serious protocol deviations will be reported to the IRB and 
CD as soon as possible and in writing not more than 7 days after the PI first learns of the 
event, unless immediate reporting is waived for specific serious adverse events as noted 
below.  Not serious unanticipated problems and not serious deviations will be reported 
to the IRB and CD as soon as possible and in writing not more than 14 days after the PI 
first learns of the event.  Written reports will be submitted in PTMS. 
 
All adverse events, deviations, and unanticipated problems will be summarized and 
reported at the time of Continuing Review. The following expected adverse events will 
not be reported at the time of Continuing Review unless they occur at a severity or 
frequency greater than expected. Adverse events occurring at a greater severity or 
frequency than expected will be reported as Unanticipated Problems. These events 
include syncope or presyncope that occur during or after blood draws that may occur up 

 
 
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to 5% of the participants, laboratory values outside the normal range with no potential 
clinical significance that may occur up to 5% of the participants, or incidental findings on 
MRI that are non-life threatening and are not requiring further medically-indicated 
diagnostic testing or treatment that may occur up to 5% of the participants.  
 
18. Alternatives to Participation 
 
Subjects do not receive any treatment in this study or forego any treatment in order to 
participate in this study. The alternative, therefore, is not to participate.  
 
19. Privacy 
 
All research activities will be conducted in as private a setting as possible. All 
interactions will take place in private settings, limiting personnel present. 
 
20. Confidentiality 
 
20. a. For research data and investigator medical records 
 
We will use hard copy case report forms to record clinical and research data, which will be 
kept in the participants’ charts.  
 
All hard copy research data and investigator medical records will be held at the nurse’s 
station during the participant’s visit and stored in a locked cabinet within a locked room 
when unoccupied. Biomarker levels, cognitive test results and MRS/fMRI images will be 
saved electronically on a secure NIA/NIH intranet with access limited to authorized NIA staff 
members and secure password protected NIA computers. All NIA members who have access 
to these databases have the proper training on participant confidentiality as well as the 
required Human Subject Protection Training. 
 
When results of an NIA/NIH research study are reported in medical journals or at 
scientific meetings, the people who take part are not named and identified. The 
NIA/NIH will not release any information about the participant’s research involvement 
without their written permission. However, if they sign a release form, for example, for 
an insurance company, the NIA/NIH will give the insurance company information from 
their medical record.  This information might affect (either favorably or unfavorably) the 
willingness of the insurance company to sell them insurance. 
 
In research collaborations all participants will be identified by a code rather than by 
their name. The results are research material and are strictly confidential. Information 
will not be released without the participant’s consent, but may be reviewed by other 
researchers for scientific purposes. 
 
All of the tests in this protocol are done for research purposes only.  It is not our policy 

 
 
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to return research results to participants, however, if we find information that is 
important to the participant’s health or well-being, we will return the results to them.  
In this event, participants will be referred to their private physician. If the participant 
requests a copy of lab results, those done under CLIA certification will be provided to 
the participant.  For tests not done in a CLIA certified lab, including genetic results, 
referrals to CLIA certified labs in the participant’s area will be provided.  We are unable 
to pay for CLIA testing.  The results or the scan from the MRI study will be provided to 
their physician with participants’ written permission.    
 
The genetic studies in this protocol are not done in a CLIA certified lab.  We test for the 
APOE ε4 gene in this study, which increases the risk of Alzheimer’s disease and 
cardiovascular disease, although it does not mean that whomever carries that gene will 
develop Alzheimer’s disease or coronary artery disease.  Participants who wish to know 
these results will meet with the principal investigator, who will consult them on the on 
the need for confirmation by a CLIA certified lab, and the significance of their particular 
genotype on AD and cardiovascular risk based on the literature.  Genetic counseling will 
be available.   
 
20. b. For stored samples 
 
Biological samples (whole blood, plasma, serum, CSF, extracted DNA and RNA) will be 
stored at the NIA Core Lab or one of our licensed facilities and released to study 
investigators and collaborators by the Principal Investigator for analysis. Participants’ names 
and identifying information will not be on the samples; samples will be identified by a 
code. The key to the code will be kept in a separate, secure area.  
 
If a participant withdraws from this research project before it is complete, any samples 
obtained before she withdraws will be kept and her privacy will be protected.  
 
20. c. Special precautions 
 
Hard copy data/records will be locked within a cabinet inside of a locked room when 
occupied. Samples without identifiers will be in a single lock container. Electronic data 
will be stored on an encrypted, password protected NIH computer or encrypted NIH 
servers. Only study investigators will have access to the samples and data, as well as the 
NIA Clinical Director and IRB monitors or auditors as part of their supervisory duties. 
 
Finally, de-identified results from clinical trials will be posted on clintrials.gov. 
 
21. Conflict of interest 
 
21. a. Distribution of NIH guidelines 
 
NIH guidelines on conflict of interest have been distributed to all investigators.   

 
 
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21. b. Conflict of interest 
 
There are no conflicts of interest to report.  
 
21. c. Role of a commercial company or sponsor 
 
There is no commercial company or sponsor for the study.  
 
22. Technology transfer 
 
The authors entered into a technology transfer agreement with Dr. Joel Kramer of the 
University of California – San Francisco for the utilization of the software EXAMINER.  
 
23. Research and Travel Compensation 
 
All participants will be compensated for their time and research-related inconveniences 
via NIA debit cards. Payments will be given as stated below. Compensation will typically 
be prorated for parts completed if the participants do not complete the study.  
Participants will be compensated for each visit as follows: 
 
Screening 
 
 
 
$50  
Baseline 
 
 
 
$350  
Week # 2 Visit  
 
 
$150  
Week # 4 Visit  
 
 
$150  
Week # 6 Visit  
 
 
$150  
Week # 8 Visit  
 
 
$500  
 
Total compensation will be $1,350 for participants that complete the study. If they do 
not complete the study or if the study physician needs to extend their participation due 
to a missed visit, they will be paid for those parts completed; blood draw $20, Mixed 
Meal Test (MMT) $50, MRI $50, lumbar puncture $150, cognitive testing $20, calorie 
restriction for one week $50, accelerometer $10/day to wear.  
 
Participants will be offered a meal on the Screening Visit, Baseline Visit, and Outcome 
Visit. 
 
No travel or lodging compensation is provided, because all subjects will be recruited 
from the greater Baltimore area.  No escort fee will be provided. 
 
 
 
 

 
 
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25. Attachments/Appendices 
 
Please see subsequent pages for attachments and appendices.

 
 
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ATTACHMENT A. Pre-Screen Checklist 
 
Calorie Restriction Checklist – Inclusion and Exclusion Criteria 
(partial) 
 
INCLUSION CRITERIA 
 
 
 BMI ≥ 27; weight £ 350 lbs 
 Age of 55-70 years 
 
EXCLUSION CRITERIA 
 
 
   History of cardiovascular disease 
   History of cerebrovascular or other neurological diseases 
   History of substance abuse in the past 6 months or positive urine drug screen 
   History of clinically significant endocrine disorders (e.g. hypothyroidism) 
   History of eating disorders, significant GI disorders or malabsorption disorders 
History of type 2 diabetes and/or use of anti-diabetes medications or insulin; 
History of hypoglycemia 
Current use of systemic corticosteroids 
Chronic HIV, HCV or HBV infections 
   Current use of immunosuppressive agents (any) 
   Contraindications for MRI (pacemakers, ferrous metal implants or shrapnel in or 
around the head, etc.) 
   Contraindications to LP, such as Coumadin, known coagulopathy. Aspirin 81 mg qd 
is allowed. Aspirin up to 325 mg qd is allowed, if withheld for 7 days prior to the LP. 
   Pregnancy or nursing. 
 
 
 

 
 
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ATTACHMENT B. ADVERTISEMENT BROCHURE AND FLIER 
 
Recruitment materials are available as separate documents. 

 
 
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ATTACHMENT C. Activity Log 
 
Activity Log (Week X) 
 
Instructions: For each day you wear the physical activity bracelet, please 
write down the times when you woke up, bathed, exercised, and went to 
bed. If one of these activities did not occur, you can leave it blank. 
 
Day 1 
 
Woke up:  
_______________________________________  
Bathed: 
 
_______________________________________ 
Exercised:  
_______________________________________ 
Went to bed: 
_______________________________________ 
 
Day 2 
 
Woke up:  
_______________________________________  
Bathed: 
 
_______________________________________ 
Exercised:  
_______________________________________ 
Went to bed: 
_______________________________________ 
 
Day 3 
 
Woke up:  
_______________________________________  
Bathed: 
 
_______________________________________ 
Exercised:  
_______________________________________ 
Went to bed: 
_______________________________________ 
 
Day 4 
 
Woke up:  
_______________________________________  
Bathed: 
 
_______________________________________ 
Exercised:  
_______________________________________ 
Went to bed: 
_______________________________________ 
 

 
 
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ATTACHMENT D. PANAS 
 
Positive and Negative Affective Schedule (PANAS) 
Intermittent Calorie Restriction 
  
Instruction:  “Do you feel this way right now?” 
 
 
 
 
Participant ID:  
 
_______________________________________________ 
Investigator/Staff Name: 
_______________________________________________ 
 
 
 

 
 
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ATTACHMENT E. Symptoms Review Questionnaire for 5-2 CR participants 
 
Symptoms Review Questionnaire 
Intermittent Calorie Restriction 
 
Since last week, on a 1-10 scale, please rate how you felt on NON-CR DAYS for: 
 
 
Since last week, on a scale from 1-10, please rate how you felt on CR DAYS for: 
 
 
Have you experienced any other symptoms that you did not have before going on the 
diet? If so, please write down those symptoms here: 
 
Participant ID:  
 
_______________________________________________ 
Investigator/Staff Name: 
_______________________________________________ 
Date:  
 
 
________________________________________________ 

 
 
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ATTACHMENT E. Symptoms Review Questionnaire for “Healthy Living Diet” participants 
 
Symptoms Review Questionnaire 
Healthy Living Diet 
 
Since last week, on a scale from 1-10, please rate these items for how you felt 
 
 
 
Have you experienced any other symptoms that you did not have before going on the 
diet? If so, please write down those symptoms here: 
 
 
 
Participant ID:  
 
_______________________________________________ 
Investigator/Staff Name: 
_______________________________________________ 
Date:  
 
 
_______________________________________________ 
 
 

 
 
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26. Consent forms 
 
Please see attached consent forms for healthy adult volunteers.