The Effect of a No-added Sugar Diet, Time-restricted Eating and Periodic Water Fasting on Long COVID Symptoms
Phase: NA
Conditions: Long Covid19, Long COVID
Sponsor: Pacific Northwest University of Health Sciences

 
 
 
DIET AND FASTING FOR LONG 
COVID; A CROSSOVER TRIAL 
7/21/2023 
Jeffery Novack PhD and Thomas Bunker PhD 
      
NCT06214455 
      

 
 
 
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Trial Protocol and Statistical Analysis Plan 
• Trial Protocol  
• Statistical Analysis Plan (page 11) 
 
Clinical trial protocol  
 
SYNOPSIS 
TITLE  
Diet and Fasting for Long COVID 
RESPONSIBILITY 
Jeffery Novack, PhD  
Principal Investigator 
Pacific Northwest University - Health Sciences 
College of Medicine, Department of biomedical Sciences 
200 University Way, Yakima WA 98901 
 
Thomas Bunker, PhD  
Co-Principal Investigator 
Unaffiliated Patient Researcher 
7665 OConnor Rd, Boulder CO 80303 
STUDY CENTER 
Pacific Northwest University - Health Sciences 
College of Medicine, Department of Biomedical Sciences 
200 University Way, Yakima, WA 98901 
PRIMARY 
OBJECTIVE 
Change in Long COVID Symptom Severity Scores and Symptoms during Treatment A 
(diet, not-fasting) vs Treatment B (diet, fasting). 
STUDY DESIGN 
Interventional, randomized, all remote, two-arm crossover study, not blinded 
STUDY ARMS 
Experimental:   Arm AB (Diet then Diet and Fasting) 
1. 2 week baseline with a low sugar diet and 10-12 hour eating window 
2. Treatment A: Eat a low sugar diet, 10-12 hour eating window for four weeks 

 
 
 
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3. Treatment B: Eat a low sugar diet, 8 hour eating window, 23 to 60 hour fast 
once a week for four weeks 
Experimental:  Arm BA (Diet and Fasting then Diet) 
1. 2 week baseline with a low sugar diet and 10-12 hour eating window 
2. Treatment B: Eat a low sugar diet, 8 hour eating window, 23 to 60 hour fast 
once a week for four weeks 
3. Treatment A: Eat a low sugar diet, 10-12 hour eating window for four weeks 
SAMPLE SIZE 
96 Long Covid subjects  (approved for 180) 
INCLUSION 
CRITERIA 
• 
Adult (18-69 years old) 
• 
Five or more common Long Covid symptoms 
• 
Free from fever > 100F and known bacterial and parasitic infections 
• 
Must indicate willingness to limit certain supplements and report all 
medications. 
• 
Must indicate willingness to make significant dietary changes - and limit daily 
eating to an 8 or 10 hour window. 
• 
Must indicate willingness to attempt 36 hr or 60 hr water fasts each week for 4 
weeks. 
• 
Have a valid email address and phone number 
• 
Reside in the United States 
• 
Be able to read and to communicate in English 
• 
Must indicate willingness to avoid "extra" supplements such as Fish Yes Oil, 
Cod liver Oil, Krill Oil, MCT oil, Coconut Oil, Turmeric/Curcumin, Berberine, 
Quercetin (> 500 mg), Red Yeast Rice, French Marine Bark extract, Red Sage, 
Ginkgo biloba, Oregano Oil, Peppermint Oil, Black seed oil, Cinnamon bark 
extract, Elderberry, Stinging Nettle, Milk Thistle, Monolaurin, Vendicinals 9, 
Tollovid, QuadraMune and all Probiotics. 
• 
Must indicate willingness to avoid certain nutraceuticals such as Zinc (more 
than 25 mg), Arginine, Glutamine, Palmitoylethanolamide (PEA), Alpha Lipoic 
Acid, L Carnitine and Taurine during the study period. 
• 
Must indicate willingness to avoid longevity supplements such as NAD+, 
Niacin, NMN, Nicotinamide Riboside, Spermidine and Fisetin during the study 
period. 
• 
Must indicate willingness to halt Olive oil consumption greater than 1 tsp daily 
during the study period. 
EXCLUSION 
CRITERIA 
• 
Likely COVID-19 or SARS-CoV-2 infection < 45 days before enrollment 
• 
Body Mass Index (BMI) must be 20 or greater 

 
 
 
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• 
Past history of an eating disorder. 
• 
Previously fasted more than 18 hours with Long COVID 
• 
Currently doing intermittent fasting 
• 
Pregnant or breast-feeding 
• 
Severe pulmonary disease requiring supplemental oxygen 
• 
Partial loss of vision due to macular degeneration 
• 
Any recent (90 days) history of malignancies, fractures, surgery, radiation, 
chemo, anesthesia, or traumas 
• 
Diagnosed with Type I or Type II Diabetes 
• 
Previous Autoimmune condition 
• 
Heart condition (Coronary artery disease, Heart valve disease, Heart No failure, 
Stroke) 
• 
Pre-pandemic Arrhythmia 
• 
Liver disease 
• 
Previous Chronic Health Conditions that did not fully resolve (Includes 
ME/CFS, Lyme Disease, and Fibromyalgia) 
• 
Prescription anticoagulation medication that cannot be halted during the study 
period. Brilinta (ticagrelor), Plavix (clopidogrel), Coumadin, Warfarin etc. 
• 
High Cholesterol medications that cannot be halted or reduced during the study 
period (Fenofibrate, Statins > 10 mg) 
• 
Anti-inflammatories or immunosuppressants such as Steroids, Low dose 
Naltrexone (LDN), Maraviroc, Remicade/Infliximab or Colchicine that cannot 
be halted during the study period 
• 
Metformin, Ivermectin, or peptides such as BPC-157 that cannot be halted 
during the study period 
SCHEDULE 
Recruiting first participant: November 2022 
Recruiting last participant: April 2023  
Study planned completion: Nov 2024 
 
BRIEF SUMMARY  
This study uses a cross-over design to test a diet change plus a 10-12 hour eating window (Treatment A) compared 
to a diet change, an 8-hour eating window and one 36-60 hour fast per week (Treatment B) in subjects screened to 
have a minimum of five long COVID-19 symptoms. Fasting is for food only, but water is available. There is a two-
week run-in with symptom surveys at the start (Week 0) and end (Week 2), followed by randomization to either 
ARM AB or ARM BA  (with weekly symptom surveys). Since the water fasts are at the beginning of the week (with 
symptoms surveyed at the end of the week) there is a 5 day washout and then a cross-over to the other treatment for 
4 weeks. 

 
 
 
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Study Hypothesis 
Time-restricted eating and a no added sugars diet decreases Long Covid symptoms and symptoms scores. The 
addition of weekly water fasts and 8 hr time-restricted eating will result in a greater decrease of Long Covid 
symptoms than 10-12 hr restricted eating and a no added sugar diet alone over a four-week period. 
Study Aims 
This study aims to: 
a)  Determine if the self-treatment of 36- or 60-hour water fasting once a week for a month reduces overall long 
COVID severity.    If so, to what degree and what is the statistical significance of the results? 
b)  Determine if eating in a 10-to-12-hour window daily and avoiding sugar for a month reduces overall long 
COVID severity.    If so, to what degree and what is the statistical significance of the results? 
c)  Determine if the self-treatment of 36- or 60-hour water fasting once a week for a month reduces the severity of 
twenty-eight common long COVID symptoms.    If so, to what degree and what is the statistical significance of the 
results? 
d)  Determine if eating in a 10 to 12 hour window daily and avoiding sugar for a month reduces the severity of 
twenty-eight common COVID symptoms.    If so, to what degree and what is the statistical significance of the 
results? 
e)  Determine which is more efficacious: "time-restricted eating, no sugar diet, and a weekly short water fast" OR 
"time-restricted eating, no sugar diet". 
Study Significance and Background: 
PASC (the long-term post-acute sequelae of COVID-19), hereafter referred to as Long COVID, is a major public 
health crisis with no proven treatment.  Estimates of the incidence of Long COVID vary from between 14 to 30% of 
those infected with SARS-CoV-2 in the initial waves of the pandemic.  About 75% of those suffering from Long 
COVID are female.   A large and variable constellation of symptoms characterizes Long COVID. The root cause of 
Long COVID is an area of active investigation.  While there is growing evidence for low-level SARS-CoV-2 viral 
persistence, there is also some evidence for persistent microclotting, lack of oxygen utilization by mitochondria 
and/or lack of oxygen uptake by tissues, various autoantibodies and EBV reactivation.  A large NIH autopsy study 
has shown that SARS-CoV-2 can infect multiple organs and tissues including the brain, heart, intestines, blood 
vessels, reproductive organs, adipose tissues, peripheral nervous tissue and eyes.  They showed that SARS-CoV-2 is 
widely distributed throughout the body, even among patients who died with asymptomatic to mild COVID-19. 
To date, the application of autophagy in human medicine has been largely stymied by the technical inability to 
measure cellular autophagy in living people.  However, there is great interest in modulating cellular autophagy to 
prevent dementias and possibly limit the development of certain cancers.  
Coronaviruses alter the two main cellular degradative processes, the ubiquitin-proteosome system and the 
autophagy-lysosome system for their own benefit.  Some viruses are able to hide from the immune system by 
suppressing antigen presentation both via the ubiquitin-proteosome-MHC class I system and via the autophagy – 
lysosome – MHC class II system.   SARS-CoV-2 does this by lysosomal targeting and degradation of MHC-I via the 
viral ORF8 protein.    Also, the ORF3a protein has been shown to block fusion of autophagosomes with lysosomes 
by destabilizing the HOPS complex Additionally, the expression of viral proteins such as envelope (E), membrane 
(M), ORF3a, and ORF7a result in an accumulation of membrane associated LC3B, a marker of 
autophagosomes.  Together, these perturbations in the autophagic flux likely promote SARS-CoV-2 replication and 
aid in evasion from immune responses. 
The investigators propose that the viral blockage of autophagosome/lysosome fusion, lysosomal degradation and 
MHC-II viral antigen presentation might be overcome with the periodic induction of cellular autophagy.   Further, 
during autophagy, viral RNA can activate Toll-Like-Receptors such as TLR7 in the lysosomes leading to the release 
of cytokines such as Interferons that indicate viral infection.    Autophagy is also known to be integral to the process 
of viral antigen presentation via MHC-II, which allows CD4+ T cells to direct immune responses (Natural Killer 
cells and Cytotoxic CD8+ T cells) towards infected cells.   Thus, it is proposed that the periodic induction of 

 
 
 
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autophagy via Intermittent fasting and short water fasts in subjects with long COVID may prime their antiviral 
immune responses and decrease their viral load. 
Many long-haulers have symptoms similar to that of Crohn’s disease and impaired autophagy in the lining of the gut 
could potentially be directly responsible.  Daily, time-restricted eating, avoiding added sugars and processed grains 
is similar to diabetic diets that strive to minimize spikes in insulin and insulin-like growth factor.   As both are pro-
growth hormones, limiting their production may help to decrease potential viral replication.   Interestingly, a study 
found that healthcare professionals following diets rich in vegetables, legumes, nuts and fish were less likely to have 
moderate or severe cases of Covid-19 than those following ketogenic or other high protein/low carbohydrate diets. 
It is entirely possible that, similar to AIDS, eventually a cocktail of antiviral drugs will be developed for SARS-
CoV-2.  Molnupiravir and Paxlovid are the first approved antivirals but are currently indicated only for early acute 
covid. To date there are no studies of fasting and its effects on long COVID.  This study could potentially point the 
way for medical researchers to study other methods of autophagy induction to treat long COVID.  If positive results 
are obtained, this study could help people suffering from “long-haul” COVID symptoms reduce or eliminate some 
of these symptoms and could lead to targeted treatments for the remaining symptoms. In addition, this is a simple 
and inexpensive treatment that most people can try if it is found to be effective in reducing symptoms. 
 
Reference Cited in Literature Review: 
Recent Advances and Future Directions.” Cancer Discovery. https://doi.org/10.1158/2159-8290.CD-19-0292. 
Chertow, Daniel, Sydney Stein, Sabrina Ramelli, Alison Grazioli, Joon-Yong Chung, Manmeet Singh, and David 
Kleiner. 2021. “SARS-CoV-2 Infection and Persistence throughout the Human Body and Brain.” Bethesda. 
Cheung, Chun Chau Lawrence, Denise Goh, Xinru Lim, Tracy Zhijun Tien, Jeffrey Chun Tatt Lim, Justina Nadia 
Lee, Benedict Tan, et al. 2021. “Residual SARS-CoV-2 Viral Antigens Detected in GI and Hepatic Tissues from Five 
Recovered Patients with COVID-19.” Gut. https://doi.org/10.1136/gutjnl-2021-324280. 
Davis, Hannah E., Gina S. Assaf, Lisa McCorkell, Hannah Wei, Ryan J. Low, Yochai Re’em, Signe Redfield, Jared 
P. Austin, and Athena Akrami. 2021. “Characterizing Long COVID in an International Cohort: 7 Months of 
Symptoms and Their Impact.” EClinicalMedicine 38. https://doi.org/10.1016/j.eclinm.2021.101019. 
Diebold, Sandra S., Tsuneyasu Kaisho, Hiroaki Hemmi, Shizuo Akira, and Caetano Reis E Sousa. 2004. “Innate 
Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA.” Science 303 (5663). 
https://doi.org/10.1126/science.1093616. 
Girdhar, Khyati, Amaya Powis, Amol Raisingani, Martina Chrudinova, Ruixu Huang, Tu Tran, Kaan Sevgi, Yusuf 
Dogus Dogru, and Emrah Altindis. 2021. “Viruses and Metabolism: The Effects of Viral Infections and Viral 
Insulins on Host Metabolism.” Annual Review of Virology. https://doi.org/10.1146/annurev-virology-091919-
102416. 
Gold, Jeffrey E., Ramazan A. Okyay, Warren E. Licht, and David J. Hurley. 2021. “Investigation of Long Covid 
Prevalence and Its Relationship to Epstein-Barr Virus Reactivation.” Pathogens 10 (6). 
https://doi.org/10.3390/pathogens10060763. 
Hannan, Md Abdul, Md Ataur Rahman, Md Saidur Rahman, Abdullah Al Mamun Sohag, Raju Dash, Khandkar 
Shaharina Hossain, Mithila Farjana, and Md Jamal Uddin. 2020. “Intermittent Fasting, a Possible Priming Tool for 
Host Defense against SARS-CoV-2 Infection: Crosstalk among Calorie Restriction, Autophagy and Immune 
Response.” Immunology Letters. https://doi.org/10.1016/j.imlet.2020.07.001. 
Heil, Florian, Hiroaki Hemmi, Hubertus Hochrein, Franziska Ampenberger, Carsten Kirschning, Shizuo Akira, 
Grayson Lipford, Hermann Wagner, and Stefan Bauer. 2004. “Species-Specific Recognition of Single-Stranded 
RNA via Till-like Receptor 7 and 8.” Science 303 (5663). https://doi.org/10.1126/science.1093620. 
Kim, Hyunju, Casey M. Rebholz, Sheila Hegde, Christine Lafiura, Madhunika Raghavan, John F. Lloyd, Susan 
Cheng, and Sara B. Seidelmann. 2021. “Plant-Based Diets, Pescatarian Diets and COVID-19 Severity: A 

 
 
 
6
 
Population-Based Case-Control Study in Six Countries.” BMJ Nutrition, Prevention and Health 4 (1). 
https://doi.org/10.1136/bmjnph-2021-000272. 
Koepke, Lennart, Maximilian Hirschenberger, Manuel Hayn, Frank Kirchhoff, and Konstantin M.J. Sparrer. 2021. 
“Manipulation of Autophagy by SARS-CoV-2 Proteins.” Autophagy. 
https://doi.org/10.1080/15548627.2021.1953847. 
Logue, Jennifer K., Nicholas M. Franko, Denise J. McCulloch, Dylan McConald, Ariana Magedson, Caitlin R. Wolf, 
and Helen Y. Chu. 2021. “Sequelae in Adults at 6 Months after COVID-19 Infection.” JAMA Network Open 4 (2). 
https://doi.org/10.1001/jamanetworkopen.2021.0830. 
Miao, Guangyan, Hongyu Zhao, Yan Li, Mingming Ji, Yong Chen, Yi Shi, Yuhai Bi, Peihui Wang, and Hong Zhang. 
2021. “ORF3a of the COVID-19 Virus SARS-CoV-2 Blocks HOPS Complex-Mediated Assembly of the SNARE 
Complex Required for Autolysosome Formation.” Developmental Cell 56 (4). 
https://doi.org/10.1016/j.devcel.2020.12.010. 
Pretorius, Etheresia, Mare Vlok, Chantelle Venter, Johannes A. Bezuidenhout, Gert Jacobus Laubscher, Janami 
Steenkamp, and Douglas B. Kell. 2021. “Persistent Clotting Protein Pathology in Long COVID/Post-Acute 
Sequelae of COVID-19 (PASC) Is Accompanied by Increased Levels of Antiplasmin.” Cardiovascular Diabetology 
20 (1). https://doi.org/10.1186/s12933-021-01359-7. 
Proal, Amy D., and Michael B. VanElzakker. 2021. “Long COVID or Post-Acute Sequelae of COVID-19 (PASC): 
An Overview of Biological Factors That May Contribute to Persistent Symptoms.” Frontiers in Microbiology. 
https://doi.org/10.3389/fmicb.2021.698169. 
Richter, Alex G., Adrian M. Shields, Abid Karim, David Birch, Sian E. Faustini, Lora Steadman, Kerensa Ward, et 
al. 2021. “Establishing the Prevalence of Common Tissue-Specific Autoantibodies Following Severe Acute 
Respiratory Syndrome Coronavirus 2 Infection.” Clinical and Experimental Immunology 205 (2). 
https://doi.org/10.1111/cei.13623. 
Singh, Inderjit, Phillip Joseph, Paul M. Heerdt, Marjorie Cullinan, Denyse D. Lutchmansingh, Mridu Gulati, 
Jennifer D. Possick, David M. Systrom, and Aaron B. Waxman. 2022. “Persistent Exertional Intolerance After 
COVID-19: Insights From Invasive Cardiopulmonary Exercise Testing.” Chest 161 (1). 
https://doi.org/10.1016/j.chest.2021.08.010. 
Singh, Komudi, Yun Ching Chen, Shahin Hassanzadeh, Kim Han, Jennifer T. Judy, Fayaz Seifuddin, Ilker Tunc, 
Michael N. Sack, and Mehdi Pirooznia. 2021. “Network Analysis and Transcriptome Profiling Identify Autophagic 
and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.” Frontiers in Genetics 12. 
https://doi.org/10.3389/fgene.2021.599261. 
Stahl, Klaus, Jan Hinrich Bräsen, Marius M. Hoeper, and Sascha David. 2020. “Direct Evidence of SARS-CoV-2 in 
Gut Endothelium.” Intensive Care Medicine 46 (11). https://doi.org/10.1007/s00134-020-06237-6. 
Uddin, Md Sahab, Anna Stachowiak, Abdullah al Mamun, Nikolay T. Tzvetkov, Shinya Takeda, Atanas G. Atanasov, 
Leandro B. Bergantin, Mohamed M. Abdel-Daim, and Adrian M. Stankiewicz. 2018. “Autophagy and Alzheimer’s 
Disease: From Molecular Mechanisms to Therapeutic Implications.” Frontiers in Aging Neuroscience. 
https://doi.org/10.3389/fnagi.2018.00004. 
Wallukat, Gerd, Bettina Hohberger, Katrin Wenzel, Julia Fürst, Sarah Schulze-Rothe, Anne Wallukat, Anne Sophie 
Hönicke, and Johannes Müller. 2021. “Functional Autoantibodies against G-Protein Coupled Receptors in Patients 
with Persistent Long-COVID-19 Symptoms.” Journal of Translational Autoimmunity 4. 
https://doi.org/10.1016/j.jtauto.2021.100100. 
Wang, X. X., B. Zhang, R. Xia, and Qiu Ying Jia. 2020. “Inflammation, Apoptosis and Autophagy as Critical Players 
in Vascular Dementia.” European Review for Medical and Pharmacological Sciences 24 (18). 
https://doi.org/10.26355/eurrev_202009_23048. 
Wodarz, Dominik, Sophie Sierro, and Paul Klenerman. 2007. “Dynamics of Killer T Cell Inflation in Viral 
Infections.” Journal of the Royal Society Interface 4 (14). https://doi.org/10.1098/rsif.2006.0195. 

 
 
 
7
 
Zhang, Yiwen, Yingshi Chen, Yuzhuang Li, Feng Huang, Baohong Luo, Yaochang Yuan, Baijin Xia, et al. 2021. 
“The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through down-Regulating MHC-Ι.” Proceedings of 
the National Academy of Sciences of the United States of America 118 (23). 
https://doi.org/10.1073/pnas.2024202118. 
  
Study's approach using a detailed, step-by-step plan indicating what will happen in your 
investigation. 
Recruitment 
1.  Recruit subjects via social media, particularly from some of the large > 10,000 member Facebook long 
COVID groups. 
2.  The Study Advertisement with basic information about the study will include a link to the initial 
interest/screening survey.   The Study flyer will also be available on a dedicated simple website with the 
url: https://longcovidtrials.info/diet-and-fasting-for-long-covid/  The website page will also contain a link 
to the initial screening symptom survey. 
Screening 
3.  Potential participants indicate interest by filling out a short interest/screening survey in REDCap and 
providing their name and e-mail address.  Participants must reside in the United States, be 18 or older, be 
younger than 70, and have at least 5 of the 28 most common long covid symptoms. 
4.  Have potential participants then fill out the Current Medications and Supplements survey and 
review both the Inclusion/Exclusion Criteria and the Informed Consent documents.  This will allow 
screening of drugs and supplements that may interfere with the protocol treatments during the Investigator 
- Participant call. 
5.  Investigator contacts the potential participants to setup a 90 min video call 
6.  Via video call, provide information and review current meds and supplements 
7.  During the video call, the Investigator goes through the Inclusion/Exclusion criteria with the potential 
subject.  If the subject meets all the criteria, then they are randomly assigned to either the ‘TRE then 
Fasting’ group or to the ‘Fasting then TRE’ group. 
      Note: Since those with long covid are predominantly female, the assignment will be random without 
regard to gender distribution. 
If the subject does not meet all the study criteria, then they are not enrolled. 
8.  Next the Investigator reviews the Participant Instructions and Study Diagram with the subject.  The 
subject is given clear instructions on what to do and when during the 10 week study.   The subject is told 
to NOT begin their First Fast until after receiving a call from one of the study medical student volunteers.
 
Further, subjects are asked to always take their Symptoms Surveys on the same day each week. 
9.  Ask subject if they have any questions 
Consent and Enrollment 
10. Next the Investigator reviews the Informed Consent with the subject within REDCap.  Ask if the 
subject has any questions. Verify the subject's identity by driver's license or some other photo 
identification with birth date (passport). 

 
 
 
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11. Verify that the subject has signed and dated the consent form while on the call.  Note: subject consent 
is being done electronically via REDCap and the consent form will be maintained within REDCap 
12.  Investigator sends subjects a PDF copy of the completed informed consent form containing electronic 
signatures of both the participant and the investigator.  This email may also include any agreement as to 
which supplements will be stopped during the 10 week study. 
Study Procedures 
13.   After enrollment, an email for the Demographics and Vaccination survey is sent to the subject. 
14.  Upon completion of the Demographics and Vaccination survey participants are sent an e-mail 
notification containing a Health Care Provider letter.  Participants are instructed to print out the letter, 
fill in the date and their name and either mail it to the Health Care Providers or hand deliver it to 
them.  The letter gives information about the study and study duration to the subject's Healthcare 
providers. 
15. The study duration is 10 weeks.        
     2 weeks Baseline:   No added sugars diet, eat in a 10 to 12 hour window daily.   Eating in a regular 
time window daily is called Time-Restricted Eating or TRE.  
If in the TRE then Fasting group: 
     4 weeks:   No added sugars diet, eat in a 10 to 12 hour window daily 
     4 weeks:   No added sugars diet, eat in an 8 hour window daily, 36 or 60 hour water fasts weekly 
If in the Fasting then TRE group: 
      4 weeks:  No added sugars diet, eat in an 8 hour window daily, 36 or 60 hour water fasts weekly    
      4 weeks:   No added sugars diet, eat in a 10 to 12 hour window daily 
A no added sugars diet means avoiding all foods with added sugars including most processed and 
packaged foods. It means avoiding all cookies, snack/candy bars, bakery goods such as pies and donuts, 
breads, tortillas, chips. However, sugars naturally present in some whole fruits are OK to eat.  
16.  For menstrual women that experience a predictable monthly flare-up of their long covid symptoms, 
ask that they wait until a few days before or a few days after their flair to take their first Baseline Long 
Covid survey.  This is to avoid a menstrual related flair-up of symptoms during the key study end-points 
at 4 weeks and 8 weeks. 
17.  Have subjects take their initial Baseline long covid symptom survey in REDCap, after 1 week they 
will take their 2nd Baseline symptom survey.  After week 2 they take their 3rd Baseline symptom survey.  
18.  REDCap will send weekly reminders to subjects concerning - when to start self-treatment A, when to 
start self-treatment B. Treatment group A, ‘TRE then Fasting’, has a low sugar diet and a 10-12 hour 
eating window. Treatment group B, ‘Fasting then TRE’, will have an 8 hour eating window with either a 
36 or 60 hour water fast once a week. 
19. FIRST FAST Subjects will be called after they complete the appropriate survey before their first water 
fast by a medical student volunteer or Dr. Jeff Novack.  They will be informed of what to expect and 
reminded to take electrolytes.  They will be reminded that they can STOP FASTING at any time.  They 
will also be reminded of how to use the symptom flare-up form (via a public link) to talk to a med student 
and potentially have Dr. Baldwin call them.  Per the new First Fast Call Medical Student Instructions, 
the medical student volunteers are expected to talk with subjects within 24 hours of receipt of a First Fast 

 
 
 
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email notification. Meanwhile, subjects are instructed not to start their first fast until after they have 
received their Pre-First Fast Call. If the medical student does not reach the subject, they leave a message 
with their cell phone number.  They then must make at least two call attempts by 4 PST the day after the 
First Fast Call notification was received.  All call attempts and their result will be documented in the 
subject’s First Fast Call for m.  As a backup, per the crossover emails, if the participant needs to start their 
first fast but has not been able to talk with a medical student by 7 pm EST, they may call Dr. Bunker.  Dr. 
Bunker will check that the subject is indeed correct to initiate their fast at this time.   He will then review 
the Fasting Safety procedures and update the First Fast Call instrument. 
20.  Survey reminders will be sent using the dateDiff function comparing today's date:time to the survey 
date of each participant's first Baseline Survey. If a subject misses a key survey, then they are considered 
withdrawn from the study. The first and third baseline surveys, the survey at the end of 4 weeks water 
fasting and the survey at the end of the relaxed time-restricted eating are considered key surveys.   
21.  If a subject wants to be removed from the study or no longer qualifies from the study, the subject will 
be removed, but the record will be maintained. 
22.  
 With successful completion of the 10 week study, participants will be sent a "Participant Completion" 
email offering a 30-45 min Study Review conference with an investigator.   As an attachment, subjects 
will get a summary of their participation in the study.  It will include their weekly Long Covid Symptom 
Survey scores and subject comments.  Also included in the "Participant Completion" email will be 
language asking the subject to not share their study Long Covid Symptom Survey scores on social 
media.   The letter will also request that subjects not share the study flier on social media - as recruiting 
for the study must be done only via investigator posts of the flier on social media. 
23. If a subject schedules a Study Review conference, at that time, a participant's results, comments, and 
adherence to the study procedures will be reviewed.   The investigator will again remind the subject to not 
share their study scores and to not share the study flyer on social media.   
Safety Procedures 
23.  During water fasts, subjects are asked to drink salt water for electrolytes twice daily.  The recipe has 
Sodium, Potassium and Magnesium in the proper ratios to replace what would normally be obtained from 
food.  This minimizes the risk of refeeding syndrome (which is already a low risk for short water fasts). ½ 
tsp Morton Lite Salt in 12 ounces of water.  In addition, participants are instructed to take a 200 mg 
Magnesium Glycinate tablet. 
24.  If experiencing severe symptoms, they are to fill out the Symptom Flare-up form via the link in their 
Participant instructions.   Their symptoms will be immediately shared with two 3rd year medical students 
via an automated REDCap e-mail. The form also provides subjects with the medical student's names and 
phone numbers.  They will try to provide 24-hour support for study subjects.  When a medical student 
identifies a safety concern, they will be referred to Dr. Mark Baldwin. Dr. Baldwin will recommend if the 
subject should contact 911 emergency immediately and/or be removed from the study. Dr. Baldwin will 
then report the removal of the subject. 
25. 
As subjects complete their weekly Symptom Surveys, REDCap notifications will check for self-reported 
Serious and Concerning Adverse Events.Dr. Baldwin, Dr. Novack and Dr. Bunker will be sent all Adverse 
Event notifications as they are reported.   The Investigator or Co-Investigator will create an Adverse 
Event instrument in REDCap within 24 hours.   They will also contact the subject if additional 

 
 
 
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information is needed.  The Investigators will leave all “medical judgment” fields blank as per the ‘Dr. 
Baldwin REDCap Instructions’. 
Dr. Baldwin will complete serious Adverse Event forms in REDCap within 48 hours of notification.  He 
will call or text the PI/Co-PI upon completion of an Adverse Event form that he deems ‘Serious’.  The 
PI/Co-PI will then report this Serious Adverse Event in IRB Mentor as soon as possible. 
For self-reported Concerning Adverse Events, Dr. Baldwin will review them weekly on Thursdays.   He 
populates all the ‘medical judgment’ fields and determines if follow-up is needed or not.     
Data Analysis 
26.  Remove identifiers from study data for any exports 
27.  Post study data to a secure SharePoint folder for analysis by our biostatisticians.  SharePoint is 
HIPAA and FERPA compliant and is encrypted at rest. 
28.  Study records will be maintained for seven (7) years. 
 
Study Protocol version 3.0 as of 7/21/2023 
 
 
 

 
 
 
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Statistical Analysis Plan 
  
Title: Diet and Fasting for Long COVID 
  
Principal Investigator: Jeffery Novack, PhD 
  
Co-Principal Investigator: Thomas Bunker, PhD 
  
Other Investigators: Mark Baldwin, DO; Robert Sorrells, PhD; Benjamin D. Horne, PhD, MStat, MPH 
  
Study Patient Advocate: Thomas Bunker, PhD 
  
Statistical Analysts: Lucy A. Horne; Robert Sorrells, PhD; Benjamin D. Horne, PhD, MStat, MPH 
  
  
1.0 Study Overview 
Introduction. Post-acute sequelae of COVID-19 (PASC), or Long COVID, is composed of a constellation of 
symptoms that arise after resolution of acute SARS-CoV-2 infection and constitutes an on-going crisis of 
international public health. Estimates of the incidence of Long COVID vary from between 6-10% in current 
literature, although this varies depending on definitions and estimates were higher in earlier phases of the pandemic. 
A majority of those reporting Long COVID are female. COVID-19 is known to directly target activated T cells and 
to inhibit or disable autophagy. The causes of Long COVID remain under investigation, but evidence suggests that 
viral persistence, persistent micro-clotting, autoantibodies, reactivation of Epstein-Barr Virus, and energy 
dysregulation may play a role. No proven treatment currently exists for Long COVID. 
Water-only fasting is reported to activate various mechanistic pathways such as autophagy, decrease excessive 
inflammation, deactivate T cells and protect activated T cells, and generally strengthen the immune system. Various 
regimens of fasting exist, with the most popular being shorter-term durations, or intermittent fasting, in which 
caloric intake ceases for 2 days or less. Intermittent fasting regimens include fasting every day for 14-16 consecutive 
hours (also known as time-restricted eating or TRE), once per week for 24 hours, two days per week on either 
consecutive or non-consecutive days, and fasting every other day (also called alternate-day fasting). Whether 
intermittent fasting reduces the severity of acute COVID-19 or PASC/Long COVID is untested but theory suggests 
that it should directly counteract many of the main adverse effects of COVID-19. 
1.1 Study Aims 
1.1.5 
Primary aim: Determine if self-treatment with 23- to 60-hour water fasting once per 
week in addition to eating in a daily 8-hour TRE window (i.e., daily 16-hour water 
fasting) and eating a low-sugar diet reduces overall Long COVID severity, as measured 
by sixty symptoms of Long COVID. 
1.2.5 
Secondary aims: 
1.2.1.5 Determine if eating in a 10- to 12-hour TRE window daily (i.e., 12- to 14-hour 
daily fasting) and eating a low-sugar diet reduces overall Long COVID severity. 
1.2.2.5 Determine if the self-treatment of 23- or 60-hour water fasting once per week 
for four weeks (in addition to 8-hour TRE and a low-sugar diet) reduces the 
severity of twenty-eight common Long COVID symptoms. 
1.2.3.5 Determine if eating in a 10- to 12-hour window daily and avoiding sugar for 
four weeks reduces the severity of twenty-eight common COVID symptoms. 
1.2.4.5 Determine which is more efficacious for reducing severity of Long COVID: a 
once-weekly 23- to 60-hour water fast with daily substantial 8-hour TRE (16-
hour fasting) and a low-sugar diet OR a daily modestly-restricted TRE and low-
sugar diet. 
2.1 Study Hypotheses 
2.1.5 
Primary hypothesis: The overall Long COVID symptom severity score is lower after 4 
weeks of once-per-week 23- to 60-hour water-only fasting [in addition to eating in an 8-

 
 
 
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hour daily TRE window (16-hour water fasting) and following a low sugar diet] than 
after 4 weeks of 10- to 12-hour daily TRE (12- to 14-hour water fasting) and a low-sugar 
diet. 
2.2.5 
Secondary hypotheses: 
2.2.1.5 The overall Long COVID symptom severity score is lower after 1, 2, or 3 weeks 
of once-per-week water-only fasting (in addition to eating in an 8-hour window 
daily and a low sugar diet) than after 1, 2, or 3 weeks of 10- to 12-hour daily 
time-restricted eating and a low-sugar diet. 
2.2.2.5 The overall Long COVID symptom severity score is lower after the initial 2-
week run-in phase of 10- to 12-hour daily TRE and a low-sugar diet reduces 
Long COVID symptom severity scores. 
2.2.3.5 The count of Long COVID symptoms is lower after 1, 2, 3, or 4 weeks of once-
per-week water-only fasting (in addition to eating in an 8-hour window daily and 
a low sugar diet) than after 1, 2, 3, or 4 weeks of 10- to 12-hour daily time-
restricted eating and a low-sugar diet. 
  
2.0 Study Population 
The trial population consists of 58 individuals, including 29 randomized to 4 weeks of once-per-week 23-60 hour 
water-only fasting plus daily 8-hour TRE (16-hour daily water fasting) and a low-sugar diet followed by 4 weeks of 
10- to 12-hour daily TRE (daily 12- to 14-hour water-only fasting) and a low-sugar diet, and the other 29 
randomized to 4 weeks of daily 10-12 hour TRE (daily 12- to 14-hour water-only fasting) and a low-sugar diet 
followed by 4 weeks of once-per-week 23-60 hour fasting plus daily 8-hour TRE (16-hour daily water fasting) and a 
low-sugar diet. All participants engaged in 10 weeks of the study protocol, including a 2-week run-in period of daily 
10- to 12-hour TRE (daily 12- to 14-hour water-only fasting) and a low-sugar diet. 
1.1 Inclusion Criteria: participants must be adults, 18 to 69 years of age; they must have 5 or more of the 
28 common Long COVID symptoms, they must reside in the United States of America, and they must 
be willing to stop the consumption of any nutritional supplements that may activate autophagy. 
2.1 Exclusion Criteria: At baseline, participants could not have a body mass index below 20 kg/m2, could 
not be pregnant or breastfeeding, and could not have had a SARS-CoV-2 infection within 45 days. 
Further, they could not carry a diagnosis of type 1 or type 2 diabetes and could not have a history of 
any eating disorder. 
3.1 Data Acquisition: All participant data were collected through pragmatic electronic means in REDCap. 
Weekly symptom and adherence data were collected through similar REDCap forms. All data were 
stored in REDCap until the end of the study enrollment and follow-up period was complete for all trial 
participants. 
  
3.0 Outcomes, Exposures, and Other Variables 
1.1 Trial Intervention and Control Arms 
1.1.5 
Active “Control” Arm: a low-sugar diet with a 10- to 12-hour daily TRE eating window 
and water-only fasting for the other daily 12 to 14 consecutive hours. 
1.2.5 
Intervention Arm: a low-sugar diet with a more restrictive 8-hour daily TRE eating 
window and 16 consecutive hours of daily fasting, along with a once-weekly 23- to 60-
hour water-only fast. 
2.1 Trial Endpoints 
2.1.5 
Primary endpoint: The primary trial endpoints are: 1) a composite of symptom severity 
scores for 28 common Long COVID symptoms and 32 less common symptoms, and 2) a 
sum of the presence or absence of the 60 symptoms. 
2.2.5 
Secondary endpoints 
2.2.1.5 A composite of symptom severity scores for 28 common Long COVID 
symptoms. 
2.2.2.5 Each of the 28 common Long COVID symptom severity scores individually. 
2.2.3.5 The count of the 28 common Long COVID symptoms that are present. 
2.2.4.5 The count of the 32 less common Long COVID symptoms that are present. 
2.2.5.5 Adherence to the assigned trial intervention. 

 
 
 
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2.3.5 
Other study data elements: percent resolution of each Long COVID symptom. 
  
4.0 Statistical Analysis Procedures 
1.1 Data Quality 
1.1.5 
Data are assessed by the principal investigators for data quality across the study and the 
investigators will keep in close contact with each participant to assess correctness of 
reporting and data capture. 
1.2.5 
Participants reporting unusual changes in symptoms will be asked to report unusual 
activities that may impact values. 
2.1 Power Calculation 
2.1.5 
As no prior study involving fasting and Long COVID exist, with the exception of two 
case reports, this trial’s sample size was estimated based on the 2022 understanding of 
Long COVID and on publications at that time about Long COVID that had a nascent 
understand of the symptoms involved in the condition. The expected effect of fasting on 
Long COVID symptoms was estimated roughly to require 40 to 90 individuals per trial 
arm, in part depending on the severity of Long COVID symptoms experienced by those 
who would enroll. 
3.1 Analysis Plan 
3.1.5 
The study population will be evaluated using an intent-to-treat approach to the statistical 
analysis. All subjects who have data regarding symptom severity scores available at each 
time point will be included in the analyses. 
3.2.5 
Demographics, anthropometric variables, risk factors, COVID-19 vaccinations, other 
medications, and socioeconomic variables will be described as percentages or means with 
standard deviations. 
3.3.5 
Symptom severity scores will be compared between the two study arms using the two-
sided paired T-test at each time point, as appropriate. Based on the randomized design of 
the study, it is expected that most if not all of these data elements will be balanced 
between the TRE arm and the Fasting arm of the study. Similar comparisons will also be 
performed to compare between the treatment arms for changes at weeks 1, 2, or 3 of each 
treatment to examine potential timing of the impact of those interventions. 
3.4.5 
The presence of individual symptoms at baseline, end of the run-in period, end of 
Fasting, end of TRE, and end of the 10-week trial will be summarized with percentages 
or means. Examination of overall changes in all participants for LC-score and 
numLCsym will use two-sided Student’s T-test to evaluate the differences in means at 
baseline and the end of the 2-week run-in phase, and at baseline compared to the final 
week of the trial. A repeated measures general linear model will be used to test 
differences between the two trial arms across baseline and the 10 end-of-week 
measurements of the LC-score and numLCsym. 
3.5.5 
Adherence to the Fasting intervention will be tracked for each participant using self-
reported assessment of whether the components of the intervention were followed each 
week of the study and will be reported as percentages. 
3.6.5 
Adverse events and major adverse events will be recorded for each participant and 
reported as percentages along with counts of events at each time point. 
3.7.5 
Analyses will use R Studio. 
3.8.5 
Exploratory analyses will examine other features of the data set 
3.9.5 
Subgroup analyses may be conducted using non-paired two-sided t-tests to identify 
statistically significant correlations between mean percent decreases in the primary 
measures and various baseline characteristics. 
 
5.0 Limitations 
1.1 Trial limitations include the lack of a parallel standard control arm, lack of randomization to just one 
treatment for each trial participant (due to the crossover design), a limited sample size, evolving 
definitions of Long COVID in the field, and a short duration of treatment by each intervention during 
the study. 

 
 
 
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2.1 Participant limitations include an open-label set of interventions conducted without blinding of 
participants to the treatment that they receive, self-selection for participation, and self-reporting of 
symptoms. 
  
6.0 References 
1.1 R Core Team (2022). R: A language and environment for statistical computing. R Foundation for 
Statistical Computing, Vienna, Austria.  URL https://www.R-project.org/.