Examining the Effects of Juice Fasting
Phase: NA
Conditions: Epigenetics, Microbiome
Sponsor: Northwestern University

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PROTOCOL TITLE:  Examining the Effects of Juice Fasting 
 
 
 
 
PRINCIPAL INVESTIGATOR: 
Melinda Ring, MD, FACP 
Executive Director 
Osher Center for Integrative Medicine at Northwestern University 
150 East Huron Avenue, Suite 1100 
Chicago, Illinois 60611 
312‐926‐6817 (Office) 
mring@nm.org 
 
Lifang Hou, MD, PhD 
Associate Professor of Preventive Medicine 
Northwestern University, Feinberg School of Medicine 
680 N Lake Shore Drive, Suite 1400 
Chicago IL 60611 
312-503-4798 (Office) 
l-hou@northwestern.edu 
 
 
Brian Joyce, PhD 
Post-Doctoral Fellow 
Northwestern University, Feinberg School of Medicine 
680 N Lake Shore Drive 
Chicago IL 60611 
312-503-5407 (Office) 
b-joyce@northwestern.edu 
 
 
VERSION NUMBER: 
1.4 

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VERSION DATE: 
05-22-18 
 
 
STUDY SUMMARY: 
 
Investigational Agent(s)  
(Drugs or Devices) 
N/A 
IND / IDE / HDE #  N/A 
Indicate 
Special Population(s) 
Children  
 Children who are wards of the state  
 Adults Unable to Consent   
 Cognitively Impaired Adults  
 Neonates of Uncertain Viability  
 Pregnant Women  
 Prisoners (or other detained/paroled individuals)  
 Students/Employees  
Sample Size 30 
Funding Source Osher Center for Integrative Medicine at Northwestern 
University 
Indicate the type of consent 
to be obtained 
Written  
Verbal/Waiver of Documentation of Informed Consent 
Waiver of HIPAA Authorization  
Waiver/Alteration of Consent Process   
Site 
 Lead Site ( For A Multiple Site Research Study) 
 Data Coordinating Center (DCC) 
Research Related 
Radiation Exposure 
Yes  
 No  
DSMB / DMC / IDMC 
Yes  
No 
 
OBJECTIVES: 
This study aims to examine the impact on epigenetic markers (methylation), gut bacteria, 
inflammation markers, measures of insulin resistance and patient reported outcomes of well- 
being in subjects on a 3-day juice-only fast vs calorie restriction vs juice added to regular diet. 
 
 
BACKGROUND: 
Food is increasingly recognized as a core component of preventive and ameliorative health 
care. An estimated 90% of cardiovascular disease and diabetes, and 70% of all cancers could 
be prevented with lifestyle measures. Juice fasting has quickly become one of the most popular 
self-prescribed dietary interventions in the United States. A wide variety of juice fasts are 
available in the popular market, the most popular variation is the three-day juice and vegetable 
fast. According to typical juice fast protocol, the participant is instructed to consume only 
fruit/vegetable juice for the duration of the fast. The purpose of this study is to assess the 
efficacy of a 3-d fruit/vegetable juice fast as a dietary intervention with the potential to impact 
epigenetic markers (methylation), increase beneficial gut bacteria, and improve measures of 

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insulin resistance leading to improved general well-being. 
 
 
Dietary interventions utilizing calorie restriction without malnutrition have supported for their 
ability to extend lifespan through reduction of metabolic rate and oxidative damage and improve 
markers of age-related diseases such as insulin resistance for diabetes since the 1930s.1  A 2010 
analysis of the mechanism of effect linked fasting to changes in IGF-1, IGFBP1, glucose, and 
insulin.2 A 30% or more decrease in circulating insulin and glucose and decline in IGF-1 can be 
observed in mammals after 3 days or more of fasting.2 Similarly, a 2016 study on 3-day 
fruit/vegetable juice fasting found significant increases in plasma and urine nitric oxide and a 
significant decrease in urinary lipid peroxidation.3 
 
 
Fruits and vegetables have also been studied for their antioxidant effects due to the phenolic 
and prebiotic compounds inherent to the foods. The phenolic and micronutrient compounds 
contribute to the effectiveness of fruits and vegetables as antioxidants. A 2013 study on the 
effect of increasing fruit and vegetable consumption found increased levels of -tocopherol, lutein, 
beta carotene, beta cryptoxanthin, and ascorbic acid through diet intervention.4 Increasing the 
amount of antioxidants in the diet can combat the effects of oxidative stress which contribute to 
the development of chronic disease. The beneficial compounds of fruits and vegetables remain 
obtainable in pressed fruit/vegetable juice. A 2017 study found pure pressed fruit and vegetable 
juices contained the necessary polyphenols and vitamins found in whole fruits and vegetables to 
maintain the positive relationship between fruit/vegetable consumption and lower incidence of 
chronic noncommunicable disease. Similar to studies with whole fruit, participants consuming 
pure fruit/vegetable juices had lower levels of lipid peroxidation and higher levels of most 
plasma/serum antioxidant vitamins.5 Therefore, pressed fruit/vegetable juices are an appropriate 
method through which to study the effects of fruit/vegetable consumption on the development of 
chronic disease. 
 
 
Despite the popularity of 3-day juice fast, there is little existing literature delineating the effects of 
the diet intervention over other fasting protocols. 3-day juice fast programs qualify as a fast 
under the definition of fasting due to the low caloric intake of the participant. The 3-day juice fast 
program we will be utilizing provides approximately 800 -900 kcal per day, 60% and 70% less 
than the average recommended daily intake for women and men age 19-30, respectively. The 
purpose of this study is to delineate the mechanism through which juice fasting interventions 
affect change in methylation, gut microbiome, and inflammation markers. 
 
 
STUDY ENDPOINTS: 
The primary endpoints of the study are changes in epigenetic markers, gut microbiota, and 
biomarkers of inflammation and insulin resistance that may potentially occur after juice fasting. 
Secondary goals of the study include providing a foundation for future studies to examine the 
mechanisms of the intervention and test the intervention in a larger scale. 

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STUDY INTERVENTIONS / INVESTIGATIONAL AGENTS: 
Group 1: Juice Fasting 
This group will be provided with the following juices, 1 of each per day, which together will 
constitute the 800-900 calorie daily diet. 
Juice Menu (Delivered by Pressed Vibrance) 
 
Name 
Calories Ingredients 
Nutrition 
Astound, 16oz 
140 
Spinach, Kale, Romaine, 
Sodium: 95mg / 4% 
Cucumber, Fennel, 
Total Carbohydrates: 34g / 
Parsley, Celery, Lemon, 
11% 
Apple, Ginger, Turmeric 
Sugar: 18g 
Protein: 4g 
Vit A: 240% 
Vit C: 190% 
Calcium: 20% 
Iron: 20% 
Delightful Greens, 
170 
Kale, Spinach, Cucumber, 
Sodium: 260mg / 11% 
16oz 
Romaine, Parsley, Celery, 
Total Carbohydrates: 30g / 
Ginger, Lemon 
11% 
Sugar: 11g 
Protein: 12g 
Vit A: 590% 
Vit C: 420% 
Calcium: 45% 
Iron: 50% 
Connective, 16oz 
120 
Aronia Berry, Spinach, 
Sodium: 110mg / 5% 
Romaine, Cucumber, 
Total Carbohydrates: 28g / 
Parsley, Lemon, Ginger 
9% 
Sugar: 15g 
Protein: 5g 
Vit A: 230% 
Vit C: 120% 
Calcium: 15% 
Iron: 25% 
Mindful, 16oz 
130 
Spinach, Kale, Romaine, 
Sodium: 250mg / 10% 
Cucumber, Parsley, 
Total Carbohydrates: 24g / 
Celery, Swiss Chard, 
8% 
Lemon, Lime, Ginger, 
Sugar: 11g 
Apple 
Protein: 8g 
Vit A: 330% 
Vit C: 200% 
Calcium: 20% 
Iron: 30% 
Nourishment, 16oz 
120 
Aronia Berry, Spinach, 
Sodium: 150mg / 6% 
Kale, Romaine, 
Total Carbohydrates: 23g / 
Cucumber, Parsley, 
8% 
Celery, Lemon, Turmeric, 
Sugar: 8g 

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Ginger, Wheatgrass, 
Broccoli Sprouts, 
Sunflower Sprouts 
Protein: 7g 
Vit A: 340% 
Vit C: 230% 
Calcium: 25% 
Iron: 30% 
Aronia Crunch, 16oz 
140 
Apple, Celery, Lime, 
Sodium: 40mg / 2% 
Aronia Berry 
Total Carbohydrates: 36g / 
12% 
Sugar: 22g 
Protein: 2g 
Vit A: 10% 
Vit C: 45% 
Calcium: 8% 
Iron: 6% 
 
Group 2: Juice + Ad Hoc Diet 
This group will be provided with the same juices as the above, but will also be instructed to 
supplement their diet as needed with their own food, without any proscribed caloric, liquid vs. 
solid, or other restrictions. 
 
 
Group 3: Caloric Restriction 
This group will be provided with the meals below to make up their 900 calorie daily diet. 
 
~900 Calorie Plant-Based Daily Meal Plan (Delivered by KitchFix) 
Calories 
Loaded Cauliflower Mash (No Bacon) + Strawberry Cashew Yogurt + Power 
Green Veggie Quinoa Bowl 
881 
Herbed Root Vegetable Side + Spice Roasted Sweet Potato Side + Paleo 
Blueberry Muffin + Broccolini 
879 
Paleo Blueberry Muffin + Loaded Cauliflower Mash (No Bacon) + Spice Roasted 
Sweet Potato Side + Power Green Veggie Quinoa Bowl 
889 
Strawberry Cashew Yogurt + Herbed Root Vegetable Side + Tomato and 
Roasted Garlic Soup 
862 
 
 
Name 
Calories 
(Calories from fat) 
Ingredients 
Nutrition 

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Loaded Cauliflower 
Mash (No Bacon), 
141.7 g 
75 
(25) 
Cauliflower, scallions, 
almond, nutritional yeast, 
parsley, thyme, 
rosemary, garlic, salt, 
black pepper 
Total Fat (g): 2.5 
Sat. Fat (g): 0.2 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 130.7 
Total Carbs (g): 12.3 
Fiber (g): 4.9 
Sugars (g): 4.2 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 4.7 
Vitamin A: 9.80% 
Vitamin C: 140.70% 
Calcium: 7.10% 
Iron: 11.70% 
Potassium (mg): 601.1 
Polyunsat Fat (g): 0 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 15.70% 
Vitamin K: 155.90% 
Thiamin: 17.30% 
Riboflavin: 15.30% 
Niacin: 8.20% 
Vitamin B6: 19.90% 
Folate: 33.40% 
Vitamin B12: 3.80% 
Pantothenic Acid: 10.80% 
Phosphorus: 8.70% 
Magnesium: 8.90% 
Zinc: 4.10% 
Selenium: 1.80% 
Copper: 5.20% 
Manganese: 17.00% 

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Strawberry Cashew 
Yogurt, 198.4 g 
453 
(292) 
YOGURT cashew, raw 
honey, lemon, coconut 
water, vanilla extract, 
psyllium, salt 
STRAWBERRY 
strawberry, lemon, 
honey, salt GRANOLA 
almonds, sunflower 
seeds, pecans, cashews, 
almond flour, maple 
syrup, clover honey, 
coconut oil, flax seed, 
vanilla extract, cinnamon, 
sea salt 
Total Fat (g): 32.4 
Sat. Fat (g): 6.6 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 81.8 
Total Carbs (g): 33.1 
Fiber (g): 5.1 
Sugars (g): 14.8 
Added Sugar (g): 7.3 
Sugar Alcohol (g): 0 
Protein (g): 12.9 
Vitamin A: 0.10% 
Vitamin C: 38.00% 
Calcium: 5.60% 
Iron: 23.70% 
Potassium (mg): 520.4 
Polyunsat Fat (g): 0 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 15.90% 
Vitamin K: 21.60% 
Thiamin: 19.20% 
Riboflavin: 8.60% 
Niacin: 5.90% 
Vitamin B6: 14.30% 
Folate: 8.30% 
Vitamin B12: 0.00% 
Pantothenic Acid: 5.50% 
Phosphorus: 34.70% 
Magnesium: 43.90% 
Zinc: 21.70% 
Selenium: 17.30% 
Copper: 60.70% 
Manganese: 59.90% 

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Power Green Veggie 
Quinoa Bowl, 326.0 g 
353 
(188) 
QUINOA quinoa, arugula, 
broccoli, asparagus, kale, 
mustard greens, parsley, 
salt SAUCE VERTE 
basil, arugula, EVO, 
garlic, red onion, lemon, 
capers, dijon, red fresno 
pepper, salt 
Total Fat (g): 20.9 
Sat. Fat (g): 2.9 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 545 
Total Carbs (g): 35.3 
Fiber (g): 7.4 
Sugars (g): 4.2 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 10.1 
Vitamin A: 106.50% 
Vitamin C: 166.30% 
Calcium: 19.70% 
Iron: 24.60% 
Potassium (mg): 789.4 
Polyunsat Fat (g): 0.1 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 28.90% 
Vitamin K: 524.40% 
Thiamin: 17.80% 
Riboflavin: 18.40% 
Niacin: 8.60% 
Vitamin B6: 23.50% 
Folate: 41.90% 
Vitamin B12: 0.00% 
Pantothenic Acid: 5.80% 
Phosphorus: 28.30% 
Magnesium: 32.60% 
Zinc: 13.90% 
Selenium: 8.70% 
Copper: 39.80% 
Manganese: 67.80% 

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Herbed Root Vegetable 
Side, 226.8 g 
222 
(34) 
Parsnip, carrot, turnip, 
redskin potato, high-oleic 
sunflower oil, parsley, 
salt, black pepper 
Total Fat (g): 3.7 
Sat. Fat (g): 0.4 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 224.6 
Total Carbs (g): 45.6 
Fiber (g): 10.3 
Sugars (g): 13.3 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 4.6 
Vitamin A: 306.60% 
Vitamin C: 83.80% 
Calcium: 10.30% 
Iron: 10.50% 
Potassium (mg): 1231.6 
Polyunsat Fat (g): 0.4 
Monounsat Fat (g): 2.6 
Vitamin D: 0.00% 
Vitamin E: 16.20% 
Vitamin K: 85.70% 
Thiamin: 16.90% 
Riboflavin: 9.20% 
Niacin: 14.80% 
Vitamin B6: 22.30% 
Folate: 27.80% 
Vitamin B12: 0.00% 
Pantothenic Acid: 12.40% 
Phosphorus: 17.80% 
Magnesium: 17.20% 
Zinc: 8.80% 
Selenium: 4.00% 
Copper: 17.80% 
Manganese: 46.40% 

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Spice Roasted Sweet 
Potato Side, 226.8 g 
229 
(20) 
Sweet potato, chili 
powder, paprika, coconut 
oil, salt 
Total Fat (g): 2.2 
Sat. Fat (g): 1.6 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 349.7 
Total Carbs (g): 66.1 
Fiber (g): 10.4 
Sugars (g): 13.7 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 5.3 
Vitamin A: 929.90% 
Vitamin C: 13.00% 
Calcium: 10.30% 
Iron: 12.70% 
Potassium (mg): 1126.7 
Polyunsat Fat (g): 0 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 7.60% 
Vitamin K: 9.70% 
Thiamin: 17.10% 
Riboflavin: 12.60% 
Niacin: 10.10% 
Vitamin B6: 35.70% 
Folate: 9.00% 
Vitamin B12: 0.00% 
Pantothenic Acid: 26.10% 
Phosphorus: 15.80% 
Magnesium: 20.90% 
Zinc: 7.00% 
Selenium: 3.30% 
Copper: 25.40% 
Manganese: 43.30% 

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Paleo Blueberry Muffin, 
62.0 g 
162 
(106) 
MUFFIN almond flour, 
coconut flour, banana, 
almond butter, almonds, 
baking powder, salt, 
baking soda, vanilla 
extract, coconut oil, 
maple syrup, coconut 
milk, pastured eggs, 
cinnamon, blueberries 
Total Fat (g): 11.7 
Sat. Fat (g): 3.3 
Trans Fat (g): 0 
Cholesterol (mg): 36.4 
Sodium (mg): 87.8 
Total Carbs (g): 13.2 
Fiber (g): 3.5 
Sugars (g): 6.9 
Added Sugar (g): 3.4 
Sugar Alcohol (g): 0 
Protein (g): 4.8 
Vitamin A: 1.60% 
Vitamin C: 3.70% 
Calcium: 5.40% 
Iron: 15.90% 
Potassium (mg): 101.8 
Polyunsat Fat (g): 0 
Monounsat Fat (g): 0 
Vitamin D: 2.00% 
Vitamin E: 52.20% 
Vitamin K: 0.10% 
Thiamin: 0.60% 
Riboflavin: 1.70% 
Niacin: 0.80% 
Vitamin B6: 2.40% 
Folate: 1.90% 
Vitamin B12: 1.50% 
Pantothenic Acid: 0.40% 
Phosphorus: 3.10% 
Magnesium: 4.30% 
Zinc: 1.60% 
Selenium: 0.20% 
Copper: 1.40% 
Manganese: 11.50% 

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Broccolini, 226.8 g 
81 
(8) 
Broccolini, garlic, salt, 
black pepper, lemon zest, 
chili flake 
Total Fat (g): 0.9 
Sat. Fat (g): 0.1 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 149.9 
Total Carbs (g): 16 
Fiber (g): 6.1 
Sugars (g): 3.9 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 6.6 
Vitamin A: 29.40% 
Vitamin C: 338.90% 
Calcium: 11.20% 
Iron: 9.60% 
Potassium (mg): 734.2 
Polyunsat Fat (g): 0.1 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 9.00% 
Vitamin K: 289.10% 
Thiamin: 11.00% 
Riboflavin: 15.90% 
Niacin: 7.40% 
Vitamin B6: 21.10% 
Folate: 35.80% 
Vitamin B12: 0.00% 
Pantothenic Acid: 13.20% 
Phosphorus: 15.30% 
Magnesium: 12.20% 
Zinc: 6.40% 
Selenium: 8.50% 
Copper: 6.10% 
Manganese: 27.90% 

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Tomato and Roasted 
Garlic Soup, 453.6 g 
187 
(57) 
SOUP tomato, vegetable 
broth, onion, red bell 
pepper, garlic, carrot, 
cashew, tomato paste, 
olive oil, basil, salt, black 
pepper, chili flakes 
GARNISH basil 
Total Fat (g): 6.3 
Sat. Fat (g): 1.1 
Trans Fat (g): 0 
Cholesterol (mg): 0 
Sodium (mg): 278.3 
Total Carbs (g): 27 
Fiber (g): 4.4 
Sugars (g): 13.8 
Added Sugar (g): 0 
Sugar Alcohol (g): 0 
Protein (g): 5.9 
Vitamin A: 114.20% 
Vitamin C: 135.90% 
Calcium: 9.50% 
Iron: 12.00% 
Potassium (mg): 693.6 
Polyunsat Fat (g): 0 
Monounsat Fat (g): 0 
Vitamin D: 0.00% 
Vitamin E: 4.00% 
Vitamin K: 25.50% 
Thiamin: 5.60% 
Riboflavin: 2.50% 
Niacin: 2.30% 
Vitamin B6: 12.20% 
Folate: 4.30% 
Vitamin B12: 0.00% 
Pantothenic Acid: 2.50% 
Phosphorus: 8.70% 
Magnesium: 9.80% 
Zinc: 5.30% 
Selenium: 4.90% 
Copper: 14.10% 
Manganese: 22.80% 
 
 
 
PROCEDURES INVOLVED: 
Up to 30 participants (15 males and 15 females) will be enrolled in the study and will be 
randomized into one of the three arms (we expect each arm will have three male and three 
female participants after attrition): the “juice fasting” arm, the “juice in addition to normal diet” 
arm, and the “caloric restriction diet” arm. Participants will come into the lab for an initial visit, 
during which they will complete questionnaires on their usual diet and demographics and will be 
informed about their assignment to one of the three arms. 
 
 
In the “juice fasting” arm, participants will be given vegetable/fruit pressed juices and be 
instructed to engage in a three-day juice fast diet totaling 800-900 kcal-per-day (nutrient 
information of juices attached). The specific juices will be assigned for each day in order to 
maintain the calorie level. In the “juice in addition to normal diet” arm, participants will be given 
the same juice for three days but continue with their usual diet in addition to the juice. For this 

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arm, there is no restriction of caloric intake or restriction to liquid only. In the “caloric restriction 
diet” arm, participants will be on a whole-food plant-based diet totaling 800-900 kcal-per-day 
(matching the daily calories of juice fasting).  
 
 
Packaged food and drink will be provided, delivered by the manufacturers (KitchFix and Pressed 
Vibrance), but participants will need to have access to their own food storage, such as a 
refrigerator. Deliveries will occur at a place and time of day specified by the participant to ensure 
delivery to the participant and immediate refrigeration, and both the participant and study 
coordinator will be notified when deliveries occur. Pressed Vibrance deliveries will be in cooler 
bags lined with ice packs on all sides, kept roughly 37-39 degrees Fahrenheit. KitchFix deliveries 
will be in cooler bags with a frozen water bottle, often delivered via refrigerated vehicle. 
 
 
In all three arms, the three-day diet intervention will be preceded and followed by three days of 
self-conducted elimination diet. The pre-intervention diet will encourage participants to eat a 
healthy, light diet for three days (basic instructions: eat only fresh raw or cooked fruits and 
vegetables (preferably organic), whole grains and eggs; drink 8 glasses of water a day; avoid or 
eliminate alcohol, caffeine, sugar, processed foods, dairy, red meat, and gluten). The post- 
intervention diet will instruct participants to gradually return to eating solid foods, and limit 
strenuous exercise (basic instructions: follow a diet similar to the pre-intervention elimination 
diet; day 1 after the intervention: eat fruits & green vegetables, and drink 8 glasses of water; day 
2 add in: nuts, gluten-free grains like whole oats, quinoa, or brown rice; day 3 add in: organic 
meats, fish, & gluten-containing grains). 
Biological samples and self-reported outcomes will be collected at four time points: baseline 
(prior to the first elimination diet period), pre-intervention (after the elimination diet and before 
the three-day intervention), post-intervention (after the intervention), and 14-day post- 
intervention. At each time point, participants will complete questionnaire on quality of life 
(PROMIS - Global Health Scale, which is an NIH measure on an individual's’ overall physical 
and psychological well-being; see appendix) and collect their own saliva and stool samples. 
Participants will be given self-administered toolkits and be taught how to collect the samples at 
their first lab visit. At each of the four time points, the research assistant will coordinate the 
collection of cheek swabs and bloodspot samples. Stool samples will be collected using a pre-
moistened wipe, with written instructions provided to participants, and both stool and saliva 
samples will be collected by the participant at home, then provided to the research assistant at 
their next visit. 
 
 
Over the course of the study, participants will be instructed to keep a diet diary days one 
through nine to record their dietary intake of each day (diet diary forms with instructions 
attached), as well as fill out a modified version of the PROMIS Global Health Scale. During the 
intervention period, the research team will also contact the participants to check in with them. 
Participants will not need to record diet data for the fourteen day post intervention period. 
 
 
At the conclusion of the study, participants will bring back the final biological samples (stool 
wipe and saliva), provide the fourth bloodspot sample, and be debriefed about the purposes of 
the study. 

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The biological samples collected will then be processed and assayed for microbiome, 
methylation, inflammatory markers, and glycemic markers. 
All questionnaire data will be stored in a REDCap database, separate from screening surveys, 
which are not to be used as study data. REDCap is a secure, web-based application designed to 
support data collection and management activities for research studies, and these databases 
will be hosted by the Feinberg School of Medicine. 
 
 
DATA AND SPECIMEN BANKING: 
If fewer participants drop out than anticipated, any excess blood/stool/saliva samples will be 
banked for later assay. All such specimens will be stored at the Center for Population 
Epigenetics. 
 
 
Consent forms and demographic information will be added to Study Tracker, per FSM 
participant tracking policy. 
 
The specimens will be stored in a -80C freezer at the Center for Population 
Epigenetics, Northwestern University, located at 680 N Lake Shore Drive, Suite 1400, 
Chicago, IL 60611. The specimen can be accessed through the principal investigators 
and lab manager. 
 
 
The procedure to release specimen and data: 
1. Requester submits application to principal investigators with specific aims and specimen 
number and list; 
2. The principal investigators review and approve the request of specimen and related 
data; 
3. The request is forwarded to lab manager, who will conduct the sample recording and 
retrieval from the freezer. 
4. The data associated with specimens related to the study including patient ID, date of 
collection, time of collection, and time point, will also be released. 
 
 
SHARING OF RESULTS WITH PARTICIPANTS: 
We have no plans to share individual participants’ results. Aggregate data will be available to 
participants upon request. 
 
 
STUDY TIMELINES: 
Recruitment and data collection will last for three months. Each participant will stay in the study 
for 21 days (if they choose to complete the study). Primary analyses are expected to be 

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completed three months later, for a total of six months. 
 
 
INCLUSION AND EXCLUSION CRITERIA: 
Participants have to meet the all of the following criteria to participate in the study: 
1. Subjects considered as healthy by the investigator based on medical history and 
completion of the screening questionnaire. 
2. Subjects who, according to the investigator, can and will comply with the requirements of the 
protocol and are available for all scheduled visits at the investigational site. 
3. Healthy male or female aged between 18 and 35 (included) years  
4. 18.5 ≤BMI ≤ 30 kg/m² 
5.   Ability to give their informed consent in writing 
 
 
Individuals meeting any of the following criteria may not participate in the study: 
 
1. Documented history of previous cardiovascular disease, including CHD (angina, 
myocardial infarction, coronary revascularization procedures or existence of abnormal Q 
waves in the electrocardiogram (EKG)), stroke, syncope, and clinical peripheral artery 
disease with symptoms of intermittent claudication. 
2. Severe medical condition that may impair the ability of the person to participate in a 
nutrition intervention study (e.g. digestive disease with fat intolerance, advanced 
malignancy, or major neurological, psychiatric or endocrine disease including diabetes). 
3. Daily use of any prescription or non-prescription medication that has a high likelihood of 
impacting systemic inflammation (e.g. non-steroidal anti inflammatories or steroids), blood 
sugar control (e.g. medication for diabetes) or the human microbiome (e.g. antibiotics). 
4. Any other medical condition thought to limit survival to less than 1 year. 
5. Known immunodeficiency disorder 
6. Illegal drug use or chronic alcoholism or total daily alcohol intake >80 g/d. 
7. Difficulties or major inconvenience to change dietary habits 
8. Impossibility to follow an elimination or juice fast diet, for religious reasons or due to the 
presence of disorders of chewing or swallowing (e.g., difficulties to consume nuts) 
9. A low predicted likelihood to change dietary habits according to the Prochaska and 
DiClemente stages of change model (Nigg, 1999). 
10. History of food allergy with hypersensitivity to any of the components of the juice or diet 
11. Patients with an acute infection or inflammation (e.g., pneumonia) are allowed to 
participate in the study 3 months after the resolution of their condition. 
12. Dietary restrictions due to medical (including allergies), religious, or other concerns 

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13. Any diagnosis of allergic rhinitis, eczema, asthma, or inflammatory bowel disease (e.g. 
ulcerative colitis or Crohn’s) from a health professional 
 
 
VULNERABLE POPULATIONS: 
This study will not include vulnerable populations (adults unable to consent, individuals who 
are not yet adults, pregnant women, and prisoners). 
 
PARTICIPANT POPULATIONS: 
 
Accrual 
Category/Group: 
Consented: 
Enrolled: 
Number: 
(Adults/Children 
Maximum Number to be 
Number to Complete 
Special/Vulnerable 
Consented or 
the Study or Needed 
Populations) 
Reviewed/Collected/Screened 
to Address the 
Research Question 
Local 
Adults 
30 
18 
Study-wide 
N/A 
N/A 
N/A 
Total: 
30 
18 
 
RECRUITMENT METHODS: 
Flyers will be posted on the Evanston campus of Northwestern University. Interested 
individuals will be directed to complete a screening questionnaire online via REDCap which will 
include questions about general health conditions (from SF-36) and demographics. Based on 
the inclusion and exclusion criteria, qualified individuals would be contacted for a phone call to 
gauge their interest in participating in the study. This data will not be used in the study – it will 
be solely for the purposes of determining eligibility, and will be housed in a separate REDCap 
database. During the phone conversation, detailed information will be given on what 
participating in the study entails, and participants will be asked at the end whether they are 
willing to participate. If the answer is yes, they will be invited for the initial lab visit during which 
hard-copy consent form will be provided. 
 
 
COMPENSATION FOR PARTICIPATION IN RESEARCH ACTIVITIES 
Participants will be compensated for their participation in the study. Each participant will be 
given a gift card for $50 when they complete the third collection of biological samples and 
surveys, and $50 when they complete the final collection of biological samples and 
surveys. 
 
 
WITHDRAWAL OF PARTICIPANTS: 
Participants will have the opportunity to voluntarily withdraw throughout the study. 
Involuntary withdrawals will be initiated when participants report experiencing an adverse 
medical event (e.g., fainting or other health effects that may be related to fasting and caloric 

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restriction). 
Failure to collect samples for one or more time periods will be reviewed on a case-by-
case basis. 
 
 
RISKS TO PARTICIPANTS: 
Risks to participants are expected to be minimal, particularly in light of the valuable 
information to be gained. 
 
 
Diet intervention: Fasting and juice cleanse diets over short periods are generally safe, but 
may cause some minor discomfort during the study period. For those on restricted intake diets, 
several minor adverse effects may occur and include constipation, headache, muscle cramps, 
diarrhea, general weakness, and rash. Most of these are short-lived, and are generally 
alleviated by adequate fluid intake and other minor diet modifications. The restricted diet may 
lead to decreases in glucose levels; this risk will be mitigated by screening participants for the 
exclusion criteria of diabetes, hypoglycemia, steroid-dependent diseases and use of 
medications that impact on blood sugar such as sulfonylureas and insulin. 
 
 
Blood Spot: There is a small risk that the puncture associated with the blood spot may cause 
bruising or infection at the site of the needle insertion. 
 
 
Weight, height and waist circumference measurements: There is no physical risk involved in 
measuring weight, height, and waist circumference but there is a risk of embarrassment or 
discomfort at being measured in one’s underclothes. 
 
 
Behavioral and psychological questionnaires: These carry a minimal risk for emotional 
discomfort and stress. 
 
 
Participants will be fully informed of all of these risks during the informed consent process and 
be given the opportunity to withdraw from the study at any time without any further obligation. 
No data will be collected until informed consent has been obtained. We expect all of these risks 
to be temporary, and to quickly subside after the study is concluded. 
 
 
Efforts to minimize risk are as follows: 
Dietary intervention: Participants will be instructed to speak to the research associate or 
principal investigator if they experience discomfort, such as constipation, diarrhea, weakness, 
muscle cramps, or dizziness. There is instruction provided with the juice cleanse on how to 
address any potential side effects stemming from the diet. Participants will be told that their 
participation is voluntary and they can choose to withdraw from the study at any time without 
any further obligation. 
 

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Blood spot: Bloodspot collection will be done as professionally and comfortably as possible. 
Blood is collected for the in-person assessments in a sterile manner to reduce the threat of 
infection. Likewise, pressure is applied to the puncture site to prevent hemorrhage and 
ecchymosis. 
 
 
Weight, height and waist circumference measurements: These measurements will be done in a 
private setting by a trained research assistant. 
 
 
Psychological questionnaires: Participants will be told that they can refuse to answer questions 
that they feel are too personal or that they feel would be too distressing to answer. 
 
 
Adverse events: Participants will be asked to report significant adverse events as soon as 
possible to study staff, and will be provided with a study phone number for this purpose. Study 
physicians will be available to study staff by pager if needed to assess possible adverse events 
and provide clinical input, including calling participants if indicated. 
 
 
POTENTIAL BENEFITS TO PARTICIPANTS: 
Participants may or may not experience health benefits from juice cleansing and/or fasting. 
Participants will receive juice or packaged caloric-restriction meals for the 3-day intervention. 
They will also be compensated for their participation in the study (up to $100). Other possible 
immediate and/or direct benefits to participants and society at large include: health and lifestyle 
changes may occur as a result of participation; knowledge may be gained about their health and 
health conditions; feeling of contribution to knowledge in the health or social sciences field. 
 
 
DATA AND SPECIMEN MANAGEMENT AND CONFIDENTIALITY: 
Biological sample collection/storage and shipment: All biological samples that will be used for 
this project will be collected and then de-identified and labeled with the following information: 
patient ID, date of collection, time of collection, and time point (baseline, pre, post and 3 days 
post). All samples will be stored in a -80C freezer in the lab located at Northwestern University, 
Department of Medical Social Sciences, 2205 Tech Drive, Suite 2-120, Evanston, IL 60208 until 
ready for transfer for laboratory analysis. 
 
 
Data handling for Methylation/Microbiome Testing 
One set of de-identified DNA samples of stool, bloodspot, cheek swab, and saliva will be 
collected in sealed plastic bags and placed with a freeze pack inside of an insulated shipping 
container. The container will be shipped to Northwestern University NUseq Core Facility, 300 E 
Superior St, Tarry 2-770, Chicago, IL 60611 for laboratory assays. 
 
 
Data handling for Glycemic and Inflammatory Biomarker Testing 

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One set of de-identified labeled bloodspot samples will be collected in sealed plastic bags and 
placed with a freeze pack inside of an insulated shipping container. We will seal the box tightly 
with tape and ship specimens via courier to: 
Laboratory for Human Biology Research at Northwestern University Department of 
Anthropology, 1810 Hinman Avenue, Evanston, IL 60208; Phone: 847-467-6965 
Contact: Dr. Thomas McDade; email: t-mcdade@northwestern.edu 
 
 
No names or individual identities will be used in publications resulting from the study. Physical 
records will be kept in an area accessible only to research staff. Research data will be stored on 
a secure, HIPAA-compliant server and drive with monitored and controlled access for study staff 
and investigators. Survey data will be hosted on secure servers. A key linking study ID to 
participant names will be kept in a password-protected file on a secure FSM server, separate 
from study data. 
 
 
Biomarker Testing  
Whole Blood Testing 
Inflammatory Stress and Glycemic Biomarkers will include (C-reactive protein, interleukin-6, 
insulin, leptin). These biomarkers will be assayed using the finger prick/filter paper collection 
procedure. Blood spot samples will be collected at each scheduled time point in the protocol at 
the Osher Center for Integrative Medicine clinic or a mutually determined region on the 
Northwestern campus (in order to accommodate participants’ schedules). Throughout all stages 
of blood collection and processing, Universal Precautions will be followed. Each participant’s 
finger will be cleaned with alcohol and then pricked with a sterile, disposable, widely available 
micro-lancet commonly used by diabetics. Five drops of whole blood will be collected on 
standardized filter paper. 
 
 
Samples will be dried overnight and stored in a -80C freezer at the Northwestern University, 
Department of Medical Social Sciences, 2205 Tech Drive, Suite 2-120, Evanston, IL 60208 until 
analysis, at which time specified samples will be transported to the appropriate lab. 
 
 
Blood spot samples will be analyzed using previously validated ELISA protocols previously 
developed for use with dried blood spots. Samples will be analyzed for inflammatory and 
glycemic index markers using a high sensitivity immunoturbidimetric assay on the Hitachi 917 
analyzer (Roche Diagnostics—Indianapolis, IN) and the Luminex® 200™ System (Luminex 
Corporation, Austin, TX), separately. All samples are run in duplicates. 
 
 
Physiologic Measures 
We will measure systolic and diastolic blood pressure, which will be measured manually using a 
stethoscope and blood pressure cuff. Participants will be assisted in sliding their left arm through 
the cuff, positioning it 0.5-inches above the elbow, and securing it with Velcro material. They will 
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also be checked manually by placing the tips of the index and second fingers of one hand on the 
inside wrist of the patient and positioning the fingers just below the base of the thumb to take the 
radial pulse at the patient’s wrist. 
 
 
Weight and waist circumference will be measured at each timepoint. Waist circumference will be 
measured according to CDC guidelines: the participant will stand, and a tape measure will be 
placed horizontally around their middle, just above the hip bones. It will be kept snug, but not 
compressing the skin, and measurement will be taken just after the participant exhales. Height 
will be measured at the initial intake. 
 
 
DNA methylation (blood) 
We will use QIAamp DNA Blood Midi Kit (Qiagen) to extract cfDNA from blood samples of study 
participants according to the manufacturer's protocol. We will then undertake unbiased, genome-
wide interrogation of DNA methylation using Illumina’s newly released Infinium Methylation EPIC 
BeadChip (EPIC array), an updated version of the widely used Illumina 450K array (terminated 
in March 2016). The EPIC array covers over 850K methylation sites across the human genome 
and includes CpG sites outside of CpG islands, non-CpG methylated sites identified in human 
stem cells, sites differentially methylated in tumor cells, FANTOM5 (Functional Annotation of 
Mammalian Genome) enhancers, ENCODE (Encyclopedia of DNA Elements) open chromatin 
and enhancers, DNase hypersensitive sites, microRNA promoter regions, as well as 93% of the 
content contained in the old Illumina 450K array. 
 
 
The Illumina Genome Studio-generated detection p-values will be used to distinguish reliable 
and unreliable probe signals, and to identify failed samples. We will remove samples that contain 
>5% failed probes at detection p>0.01, as well as CpG sites that failed in >5% of samples (i.e., 
calling rate <95%). CpG probes mapped to ambiguous locations in the human genome will be 
detected and removed. To avoid potential SNP-in-probes bias, we will also exclude CpG probes 
containing common SNPs (MAF [minor allele frequency] >0.01) based on dbSNP. The final 
analysis set will include only CpG probes located on autosomes. The level of methylation, 
represented by the ratio of methylated signal to total signal and the M-value (log2 ratio of 
methylated signal over unmethylated signal) will be determined for each interrogated locus. We 
will also conduct analyses using the β-value (i.e., the proportion of modified signal in total signals 
from 0 to 1), which has a more straightforward biological interpretation. The methylation levels 
will be quantile-normalized across all samples. Potential batch effects and blood cell 
subpopulation proportion estimated by Houseman’s method 41 will be corrected using 
ComBat.42 To account for multiple comparison issue, we may 1) narrow down the genome- wide 
scan (~300,000 unambiguous autosomal CpGs) to those well-annotated genic CpG sites 
(~100,000); and 2) further narrow down to highly variable CpG sites, for example top 10% of 
highly variable (informative) CpGs (~10,000 CpGs) across the samples. 
 
DNA extraction, 16S rRNA sequencing and microbiome analysis 
Saliva samples and buccal cell DNA will be extracted using QIAamp DNA Mini Kit (Qiagen). Stool 
sample DNA will be extracted using QIAamp DNA Stool Mini Kit (Qiagen). The quality and 
quantity of the DNA will be examined using Nanodrop spectrophotometer (ThermoFisher 
Scientific). 16S rRNA amplicon library will be prepared using Nextera XT DNA Library Preparation 
Kit (Illumina) following the manufactures protocol. DNA (100 ng) will be used as template for PCR 

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to amplify the 16S rRNA region. The 16S rRNA amplicon libraries will then be sequenced on a 
MiSeq system (Illumine). For the 16S rRNA amplicon analysis, the individual library read files will 
be paired and combined in silico (16S–total) and analyzed for taxonomic annotation using the 
RDP tool. 
 
 
PROVISIONS TO MONITOR THE DATA TO ENSURE THE SAFETY OF PARTICIPANTS: 
The study poses slight risk to participants. The research team will contact each participant each 
day during the 3-day intervention period to check on their well-being and to monitor for adverse 
events relating to the diet. Possible mild side-effects include headaches, fatigue, difficulty 
thinking, moodiness, stomach pain and hunger pangs, and changes in bowel function or 
frequency of urination. For any participant feeling poorly, the research team member will 
encourage the participant to stay hydrated, and pass along a report immediately to the PI. The PI 
will be the primary individual responsible for safety monitoring. If necessary, the participant will 
be encouraged to cease the diet and resume their usual intake. Any significant adverse side 
effects including syncope (fainting) or dehydration will lead to immediate cessation of the 
intervention for that participant. 
The research team will review cumulative data from patient feedback on a weekly basis to 
monitor for adverse effects. No outcomes bad enough to warrant termination of the research 
study are expected. However, it will be on an individual basis that this option will be considered. 
 
 
PROVISIONS TO PROTECT THE PRIVACY INTERESTS OF PARTICIPANTS: 
We will collect no identifying information with the data of interest to this study. All such de- 
identified data will be stored on a secure, password-protected computer at the Osher Center for 
Integrative Medicine or the Department of Preventive Medicine. Only the study research 
assistant and principal investigators will have access to these data. The trained research 
assistant will explain all study procedures in detail during the informed consent process and 
answer any of the participants’ questions. Participants’ names and mailing addresses will be 
provided to KitchFix or Pressed Vibrance for the purposes of food/drink delivery only. This 
information will not be stored with any study data. 
 
 
Encrypted and computerized data of the laboratory measurement will be used. Throughout the 
conduct of the present study, stringent guidelines will be used to ensure privacy and 
confidentiality. The methylation data generated from this project will be stored without the 
original identifying information in the Department of Preventive Medicine at Northwestern 
University’s Feinberg School of Medicine where all data entry and data management will be 
performed. Double entry with 100% verification will be used to ensure accuracy of the process. 
 
 
COMPENSATION FOR RESEARCH-RELATED INJURY: 
If any participant appears to become ill or get injured as a result of this study, they will be 
encouraged to seek medical treatment through their doctor or treatment center of choice. They 
will be told that they should promptly to tell the PI and research team about any illness or injury. 
 
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participation in this research study. This does not keep a participant from seeking to be paid 
back for care required because of a bad outcome. 
 
 
ECONOMIC BURDEN TO PARTICIPANTS: 
This study is not expected to present economic burden to participants. The study will assume 
the full costs of all special diets used. In addition, the study will take place on Northwestern 
campus and so it should not incur additional transportation cost for Northwestern students. 
 
 
CONSENT PROCESS: 
Individuals who have completed the screener and who are deemed eligible for the study will be 
contacted for a phone call. During the phone call, specifics of the study will be described to 
participants. If they are interested in participating in the study, they will be invited for a lab visit, 
during which a written informed consent document will be provided. Participants will be given 
time to read the document and a member of the research team will be available to answer any 
questions they might have. Participants will then be given a private space to decide if they want 
to participate in the study, and if so, sign on the informed consent. 
 
 
Hard-copy informed consent will be provided for the participants to sign during their initial lab 
visit. The research team will keep the signed documents and offer to make a copy for the 
participants. 
 
 
Non-English Speaking Participants 
Due to staff limitations, only English-speaking participants will be enrolled in this study. 
 
 
QUALIFICATIONS TO CONDUCT RESEARCH AND RESOURCES AVAILABLE: 
The study procedures will be conducted with a trained research assistant experienced and 
certified in human subjects research and privacy protections (including HIPAA). It will be 
supervised by the three PIs, who between them have many years of experience conducting 
human subjects research. With a small total sample size, recruitment via on-campus 
advertisements should be achievable in the desired timeframe. Assuming 50% of participants 
are ineligible or withdraw, we will need access to 36 participants. Participants will be recruited 
from the multitude of education programs across the Evanston Northwestern campus; 
Northwestern University is a private institution with a total undergraduate enrollment of 8,353. 
 
 
The research assistant will be responsible for collecting biospecimen and questionnaire data, 
and conducting daily follow-up phone calls to monitor participant adherence to the diets of 
interest. PIs will supervise the study conduct and data collection, monitor potential adverse 
events, and assist with questions as needed. A trained research assistant will conduct the 
bloodspot collection and physiologic measurements in a private setting at the Northwestern 
Medicine Osher Center for Integrative Medicine, located at 150 E. Huron Avenue, Suite 1100, 
Chicago, IL 60611, or a mutually determined private space on campus. 

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The research assistant and PIs will review the approved study protocol together, along with all 
consent forms, scripts, and other participant materials to ensure agreement. 
 
 
Participants will be asked to report significant adverse events as soon as possible to study staff, 
and will be provided with a study phone number for this purpose. Study physicians will be 
available to study staff by pager if needed to assess possible adverse events and provide clinical 
input, including calling participants if indicated. 
 
 
All persons assisting with the research will participate in relevant weekly or biweekly team 
meetings (in person or virtual) to remain adequately informed about the protocol, the research 
procedures, and their duties and functions during recruitment, implementation and data 
analysis. 
 
 
The study will take place at the Evanston and Chicago campuses of Northwestern University. 
 
 
Bloodspot collection and physiologic measurements will be performed in a private setting at the 
Northwestern Medicine Osher Center for Integrative Medicine, located at 150 E. Huron Avenue, 
Suite 1100, Chicago, IL 60611, or a mutually determined private space on campus. 
 
 
DNA extraction will be performed at 710 N Fairbanks Ct, Olson 8-360, Chicago, where the lab 
for the Center for Population Epigenetics is located. The lab space and equipment (including 
centrifuge, thermal cycler, Nanodrop spectrometer, refrigerator, freezer) can be used for DNA 
extraction, concentration measurement and sample storage. 
 
 
Biomarker extraction will be performed at Laboratory for Human Biology Research at 
Northwestern University Department of Anthropology, 1810 Hinman Avenue, Evanston, IL 
60208. 
 
 
STUDY-WIDE RECRUITMENT METHODS: 
N/A 
 
 
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physiological, psychological, and behavioral outcomes. Antioxidants and Redox 
Signaling, 14(2), pp. 275-287. doi: 10.1089/ars10.3253. 
2. Fontana, L., Partridge, L., Longo, V.D. (2010) Extending healthy lifespan—from yeast to 
humans. Science, 328, pp. 321–326. 

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3. Henning, S.M., Shao, P., Qing-Yi, L., Yang, J., Huang, J., Ru-Po, L., Thames, G., Heber, D., & 
Li, Z. (2016). Health effects of 3-day fruit and vegetable juice fasting. Advanced Nutrition, 7(48A). 
4. Rink, S. M., Mendola, P., Mumford, S. L., Poudrier, J. K., Browne, R. W., Wactawski- Wende, J., 
Schisterman, E. F. (2013). Self-report of fruit and vegetable intake that meets the 5 a day 
recommendation is associated with reduced levels of oxidative stress biomarkers and increased 
levels of antioxidant defense in premenopausal women. Journal of the Academy of Nutrition and 
Dietetics, 113(6), 776-785. doi:10.1016/j.jand.2013.01.019 
5. Zheng, J., Zhou, Y., Li, S., Zhang, S., Zhou, T., Dong-Ping, X., & Hua-Bin, L. (2017). Effects 
and mechanisms of fruit and vegetable juices on cardiovascular disease. International 
Journal of Molecular Sciences, 18(3), 555; doi:10.3390/ijms18030555.