This study presents a comprehensive deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS), revealing key clinical and biological features. PI-ME/CFS is characterized by a distinct effort preference, rather than physical or central fatigue, due to dysfunction in integrative brain regions potentially linked to central catechol pathway dysregulation, affecting autonomic function and physical conditioning. Immune profiling showed chronic antigenic stimulation with increased naive and decreased switched memory B-cells. Gene expression and metabolic pathway alterations were consistent with cellular phenotypic studies and varied by sex. These findings suggest unique insights into the pathophysiology of PI-ME/CFS, which may guide future interventions. ME/CFS is a disabling disorder with persistent fatigue, exercise intolerance, and cognitive symptoms, but the underlying mechanisms remain unclear. Previous studies have reported immunologic, bioenergetic, and physiological alterations, but results are inconsistent. There are currently no effective treatments for ME/CFS, and developing new treatments is hindered by the difficulty in defining cases or tracking responses through symptoms or biomarkers. A major challenge in ME/CFS research is case assessment due to the absence of a diagnostic biomarker. Over 20 diagnostic criteria highlight the difficulty in defining the clinical symptoms of ME/CFS. The symptoms are non-specific and overlap with other diseases, leading to frequent misattribution. ME/CFS often occurs after an acute infection, designated post-infectious ME/CFS (PI-ME/CFS), with an estimated incidence of 10–12% after certain infections. The most well-known association of PI-ME/CFS has been with the Epstein-Barr Virus, but as the full extent of the sequelae of the COVID-19 pandemic are better understood, SARS-CoV-2 may become an even stronger correlate. In 2016, the National Institutes of Health (NIH) launched an initiative to study ME/CFS. The NIH Division of Intramural Research developed an exploratory clinical research program to perform deep phenotyping on a cohort of PI-ME/CFS participants and healthy volunteers (HV) as controls. Prior to the SARS-CoV-2 pandemic, this study recruited a cohort of well-characterized PI-ME/CFS patients and applied modern broad and deep scientific measures to describe their biophenotype compared to HVs. The aim was to identify relevant group differences that could generate new hypotheses about the pathogenesis of PI-ME/CFS and provide direction for future research. Over 75 scientists and clinicians across 15 of the 27 institutes that comprise the NIH contributed to this multi-disciplinary work. Importantly, we developed rigorous inclusion criteria which comprised detailed medical and psychological evaluations to minimize diagnostic misattribution. A relatively homogenous population was recruited in whom symptoms were initiated after infection. This study aimed to investigate the underlying pathophysiologicalThis study presents a comprehensive deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS), revealing key clinical and biological features. PI-ME/CFS is characterized by a distinct effort preference, rather than physical or central fatigue, due to dysfunction in integrative brain regions potentially linked to central catechol pathway dysregulation, affecting autonomic function and physical conditioning. Immune profiling showed chronic antigenic stimulation with increased naive and decreased switched memory B-cells. Gene expression and metabolic pathway alterations were consistent with cellular phenotypic studies and varied by sex. These findings suggest unique insights into the pathophysiology of PI-ME/CFS, which may guide future interventions. ME/CFS is a disabling disorder with persistent fatigue, exercise intolerance, and cognitive symptoms, but the underlying mechanisms remain unclear. Previous studies have reported immunologic, bioenergetic, and physiological alterations, but results are inconsistent. There are currently no effective treatments for ME/CFS, and developing new treatments is hindered by the difficulty in defining cases or tracking responses through symptoms or biomarkers. A major challenge in ME/CFS research is case assessment due to the absence of a diagnostic biomarker. Over 20 diagnostic criteria highlight the difficulty in defining the clinical symptoms of ME/CFS. The symptoms are non-specific and overlap with other diseases, leading to frequent misattribution. ME/CFS often occurs after an acute infection, designated post-infectious ME/CFS (PI-ME/CFS), with an estimated incidence of 10–12% after certain infections. The most well-known association of PI-ME/CFS has been with the Epstein-Barr Virus, but as the full extent of the sequelae of the COVID-19 pandemic are better understood, SARS-CoV-2 may become an even stronger correlate. In 2016, the National Institutes of Health (NIH) launched an initiative to study ME/CFS. The NIH Division of Intramural Research developed an exploratory clinical research program to perform deep phenotyping on a cohort of PI-ME/CFS participants and healthy volunteers (HV) as controls. Prior to the SARS-CoV-2 pandemic, this study recruited a cohort of well-characterized PI-ME/CFS patients and applied modern broad and deep scientific measures to describe their biophenotype compared to HVs. The aim was to identify relevant group differences that could generate new hypotheses about the pathogenesis of PI-ME/CFS and provide direction for future research. Over 75 scientists and clinicians across 15 of the 27 institutes that comprise the NIH contributed to this multi-disciplinary work. Importantly, we developed rigorous inclusion criteria which comprised detailed medical and psychological evaluations to minimize diagnostic misattribution. A relatively homogenous population was recruited in whom symptoms were initiated after infection. This study aimed to investigate the underlying pathophysiological