Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, debilitating disease with inconclusive causes and no laboratory-based diagnostic tests. Oxygen tension (PO2)-regulated red blood cell (RBC) capillary velocity has emerged recently as a new mechanism regulating cerebral capillary perfusion and measures RBC responses to local hypoxia. Such intrinsic RBC responses to PO2 changes may change distinctly under different pathological conditions and thus represent a new RBC-based approach for disease diagnosis. Indeed, our preliminary results showed that RBCs collected from ME/CFS patients exhibited impaired velocity in a microfluidic capillary in response to reduced PO2 and PO2-regulated RBC capillary velocity was improved significantly when patients received craniocervical instability surgery, strongly suggesting that PO2-regualted RBC capillary velocity may represent a new characteristic of ME/CFS that might be used to diagnose ME/CFS and measure ME/CFS progression. Here, we extend our preliminary study and propose an interdisciplinary approach combing microfluidics, machine learning and RBC cytokine assay to examine rigorously the accuracy of PO2-regulated RBC capillary velocity as a new laboratory test for ME/CFS and investigate mechanistically the roles of RBC cytokine signaling in ME/CFS. Aim 1 will measure PO2-regulated RBC capillary velocity using RBCs from 96 participants and compare the results of ME/CFS patients with age, gender and race-matched healthy controls. Furthermore, we will develop machine-learning algorithms to establish a diagnostic classifier to validate PO2-regulated RBC capillary velocity as a new laboratory test for ME/CFS and assess its feasibility to differentiate ME/CFS patients with different disease severity. Mechanistically, we will examine hemoglobin-band 3 interactions in RBCs from ME/CFS patients and examine their correlations with increased oxidative stress in ME/CFS. Aim 2 will quantify the cytokine profile in RBCs from ME/CFS patients and compare them with age, gender and race-matched healthy controls. Furthermore, we will alter the RBC cytokine profile and measure the corresponding changes of PO2-regulated RBC capillary velocity to examine whether RBC cytokine profile plays a role in the modulation of PO2-regulated RBC capillary velocity. These experiments will provide a previously unrecognized RBC cytokine signaling in ME/CFS and add new insights to the immune dysregulation in ME/CFS. Together, the proposed studies exploit new tools and technology to develop a new laboratory test for ME/CFS and reveal mechanistims underlying PO2-regulated RBC capillary velocity in ME/CFS, which we believe will directly improve the diagnosis and treatment of ME/CFS.