Background Long COVID syndrome (LCS) is a heterogeneous clinical condition that develops
after acute SARS-CoV-2 infection, affecting at least 10% of patients. LCS is
characterized by persistent symptoms lasting longer than 2-3 months following a confirmed
SARS-CoV-2 infection. The most disruptive symptoms include fatigue, shortness of breath,
pain, depression/anxiety, and cognitive impairment, significantly impacting daily
activities, income, and quality of life.

Pathophysiology of LCS

Immunological Dysregulation:

Persistent activation of monocyte populations, including resident and infiltrating
macrophages, dendritic cells, and neutrophils, potentially causing tissue damage.

Reservoirs of SARS-CoV-2 may persist, particularly in respiratory and gastrointestinal
tracts, contributing to ongoing lymphocyte activation.

Autoimmunity involving T-cells and B-cells has been observed, with cytokines such as
interferon gamma, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-17 frequently implicated.

Tissue fibrosis, a consequence of inflammation, is commonly reported.

Vascular Dysfunction and Coagulopathy:

Impaired fibrinolysis and platelet hyperactivation contribute to microclot formation,
entrapping pro-inflammatory molecules like IL-6.

Platelet-poor plasma in LCS patients shows high levels of fibrin amyloid microclots,
impairing microcirculation and exacerbating immunopathology.

Aberrant immune cell activity increases thrombosis through autoantibodies and NETosis.

Persistent endothelial dysfunction is a common feature, contributing to numerous LCS
symptoms.

Gastrointestinal Involvement:

SARS-CoV-2 infection reduces intestinal microbiota diversity, with recovery correlating
to reduced LCS risk.

Specific bacterial taxa, such as Escherichia, are associated with severe COVID-19 and
LCS.

Functional dyspepsia and irritable bowel syndrome, related to microbial dysbiosis, are
common post-COVID conditions.

Gut microbiota influences LCS-related biological functions, particularly psychological
symptoms, through modulation of brain chemicals and gut-blood barrier disruption.

Potential of Fasting as a Treatment for LCS Fasting shows promise as a treatment for LCS
by modulating the same cellular systems affected by LCS. A recent small study indicated
that 92% of LCS patients experienced symptom improvement during long-term fasting, with
notable effects on circulating inflammatory molecules, C-reactive protein levels, and
dyslipidemia. Fasting can increase beneficial gut microbial genera and alter blood
metabolites, potentially disrupting dysfunctional circuits in LCS.

Significance LCS affects over 150 million people worldwide, with more than half
experiencing significant work and lifestyle disruptions. Effective, low-cost, and
attainable therapeutic options are urgently needed. Medically supervised fasting,
particularly intermittent fasting, could be an accessible and safe intervention with
minimal physician oversight and low malnutrition risk. Fasting may also prevent other
chronic illnesses associated with LCS, such as type 2 diabetes mellitus.

Hypothesis Our primary hypothesis is that ambulatory fasting intervention using the
Buchinger-Wilhelmi method is a feasible treatment for LCS. We propose that medically
supervised long-term fasting will improve perceived health and quality of life in LCS
patients by disrupting inflammatory immuno-metabolic feed-forward loops, thus
ameliorating immune and vascular pathologies.

Methodology and Analysis Patient Eligibility

Inclusion Criteria:

Age 18-64 Diagnosis of LCS (post-acute COVID-19 symptoms persisting ≥12 weeks) Normal BMI
(18.5 to 25 kg/m²) Marginal iron status (PF < 25 ng/ml) Ability to communicate in and
comprehend English, German, or French Written and signed consent Willingness to consent
to specimen collection and use

Exclusion Criteria:

BMI < 18.5 kg/m² or significant recent weight loss History of eating disorders within the
past five years Severe internal disease or chronic inflammatory illness other than LCS
Participation in another intervention study Recent fasting or vegan diet Pregnancy or
breastfeeding Existing ME/CFS or early autonomous dysfunction Chronic inflammatory bowel
diseases, celiac disease, or colorectal cancer Use of anti-psychotic drugs or recent
antibiotic use Contraindication for additional blood draws Start of novel drug therapy
Intervention

The primary goal is to perform a single period of periodic fasting (PF) using the
Buchinger procedure, involving an initial bowel cleanse followed by a 7-day fasting
period. The fasting regimen includes:

Preparation Phase: Three days of relief with reduced food intake, avoiding stimulants.

Fasting Week: A laxative fluid intake on the first day, followed by 7 days of a dietary
energy supply of a maximum of 350 kcal per day (vegetable broths and juices), and
consumption of calorie-free water or tea.

Support and Guidance: Detailed instructions, face-to-face consultations, and digital
application (MyCap) for adherence confirmation via urine ketone measurements.

Sample and Data Collection Sample Size: 20 patients for the pilot study to test
feasibility.

Study Outline:

Inclusion visit for risk explanation, medical history, and physical examination.

Daily follow-up via telephone and online appointments during the fasting phase.
Questionnaire-based assessment via MyCap Smartphone Application.

Parameters to be Quantified:

Blood samples for safety parameters, metabolites, immune activity indicators, and
additional analyses.

Stool samples for microbiome composition and activity. Urine samples for metabolomics.
Saliva samples for cortisol measurements. Analysis and Results Interpretation Results
will be analyzed descriptively, with means, standard deviations, and graphs summarizing
data. Exploratory analysis will identify patterns, trends, or unexpected results.
Correlation with improvement in biomedical parameters will be assessed, with a larger
study needed for establishing causal links.

Risks Blood Draw: Discomfort, bleeding, swelling, pain, rare nerve damage, or infection
at the injection site, and fainting risk.

Periodic Fasting Protocol: Reduction in blood pressure, closely monitored with
non-medical interventions if necessary.

Data Collection and Storage Data is collected using REDCap, a secure, web-based software
platform, with pseudonymized data stored for at least 10 years post-study. The Principal
Investigator ensures compliance with national laws and GDPR.

Regulatory and Ethical Considerations Informed Consent: Participants receive an easily
understood Participant Information Sheet and Informed Consent Form, with contact
information for study personnel.

Confidentiality and Privacy: Compliance with national laws and GDPR, with pseudonymized
data transfer to third parties only with participant consent.

Financing and Insurance: Supported by the Direction de la Santé, CHNP, and the University
of Luxembourg.

Conclusion This pilot study aims to test the feasibility of using the Buchinger-Wilhelmi
fasting method for treating LCS. If successful, fasting could provide a low-cost,
accessible, and effective therapeutic option for LCS sufferers worldwide.