This study aimed to conduct deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) to better understand its pathophysiology. PI-ME/CFS is a disabling disorder with poorly defined clinical phenotypes and no effective treatments. The study recruited 17 PI-ME/CFS participants and 21 healthy controls (HV) using rigorous criteria. Key findings include: 1. **Effort Preference**: PI-ME/CFS participants showed altered effort preference, preferring to avoid harder tasks, likely due to central catechol pathway dysregulation affecting autonomic functioning and physical conditioning. 2. **Autonomic Dysfunction**: PI-ME/CFS participants had diminished heart rate variability, altered frequency domain differences, and decreased baroreflex-cardiovascular function, suggesting altered autonomic tone. 3. **Motor Performance**: Despite normal grip strength, PI-ME/CFS participants showed impaired sustained effort and motor performance, with a lower decline in button-press rate over time, indicating pacing to limit exertion. 4. **Cerebrospinal Fluid Analysis**: PI-ME/CFS participants had decreased levels of dopamine-related metabolites and catechols, with correlations observed between these metabolites and cognitive symptoms. 5. **Immune Profiling**: PI-ME/CFS participants showed increased immune activation, with elevated PD-1+ CD8+ T-cells in cerebrospinal fluid and sex-based differences in immune cell subpopulations. 6. **Gene Expression and Metabolism**: Gene expression profiles and metabolic pathways in peripheral blood mononuclear cells and muscle tissue revealed sex-based differences, with distinct biological processes perturbed in male and female PI-ME/CFS patients. 7. **Microbiota**: Stool samples showed significant differences in microbial community composition between PI-ME/CFS and HV groups. 8. **Longitudinal Follow-Up**: Four out of 17 PI-ME/CFS participants experienced spontaneous full recovery within four years, while no new medical diagnoses were reported. This comprehensive study provides insights into the underlying pathophysiology of PI-ME/CFS, highlighting the importance of effort preference, autonomic dysfunction, and immune activation in the disease's progression. These findings may guide future research and interventions.This study aimed to conduct deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) to better understand its pathophysiology. PI-ME/CFS is a disabling disorder with poorly defined clinical phenotypes and no effective treatments. The study recruited 17 PI-ME/CFS participants and 21 healthy controls (HV) using rigorous criteria. Key findings include: 1. **Effort Preference**: PI-ME/CFS participants showed altered effort preference, preferring to avoid harder tasks, likely due to central catechol pathway dysregulation affecting autonomic functioning and physical conditioning. 2. **Autonomic Dysfunction**: PI-ME/CFS participants had diminished heart rate variability, altered frequency domain differences, and decreased baroreflex-cardiovascular function, suggesting altered autonomic tone. 3. **Motor Performance**: Despite normal grip strength, PI-ME/CFS participants showed impaired sustained effort and motor performance, with a lower decline in button-press rate over time, indicating pacing to limit exertion. 4. **Cerebrospinal Fluid Analysis**: PI-ME/CFS participants had decreased levels of dopamine-related metabolites and catechols, with correlations observed between these metabolites and cognitive symptoms. 5. **Immune Profiling**: PI-ME/CFS participants showed increased immune activation, with elevated PD-1+ CD8+ T-cells in cerebrospinal fluid and sex-based differences in immune cell subpopulations. 6. **Gene Expression and Metabolism**: Gene expression profiles and metabolic pathways in peripheral blood mononuclear cells and muscle tissue revealed sex-based differences, with distinct biological processes perturbed in male and female PI-ME/CFS patients. 7. **Microbiota**: Stool samples showed significant differences in microbial community composition between PI-ME/CFS and HV groups. 8. **Longitudinal Follow-Up**: Four out of 17 PI-ME/CFS participants experienced spontaneous full recovery within four years, while no new medical diagnoses were reported. This comprehensive study provides insights into the underlying pathophysiology of PI-ME/CFS, highlighting the importance of effort preference, autonomic dysfunction, and immune activation in the disease's progression. These findings may guide future research and interventions.