Background: Post-COVID-Syndrome (PCS) is the term for a condition with persistent symptoms in a proportion of COVID-19 patient after asymptomatic, mild or severe disease courses. Numbers vary but the current estimate is that after COVID-19 approximately 10% develop PCS. The aim of our study was to evaluate the impact of SARS-CoV-2 infection on the gastrointestinal (GI) tract and associations with the development of PCS with fatigue, post-exertional malaise (PEM), orthostatic dysregulation, autonomous dysregulation and/or neurocognitive dysregulation. Methods: By combining medical record data from a prospective observational study with symptom analysis before, during, and after SARS-CoV-2 infection, we aimed to identify potential risk factors and predictive markers for PCS. Additionally, we analyzed blood, saliva, and stool samples from this well-characterized PCS patient cohort to biologically validate our findings. Results: We identified significant associations between pre-existing GI complaints and the development of PCS Fatigue. PCS patients showed higher LBP/sCD14 ratios, lower IL-33 levels, and higher IL-6 levels compared to control groups. Our results highlight the critical role of the GI tract in PCS development of post-viral Fatigue. Conclusion: We propose that the viral infection disrupts pathways related to the innate immune response and GI barrier function, evidenced by intestinal low-grade inflammation and GI barrier leakage. Monitoring GI symptoms and markers before, during and after SARS-CoV-2 infection is crucial for identifying predictive clinical phenotypes in PCS. Understanding the interaction between viral infections, immune responses, and gut integrity could lead to more effective diagnostic and treatment strategies, ultimately reducing the burden on PCS patients.