Background Erythrocytes and the apical surface of vascular endothelial cells are covered by a dense sheet of glycans termed the glycocalyx. It is composed of a negatively charged network of proteoglycans, glycoproteins, and glycolipids. The negative charge is afforded and maintained through heavy sialylation and sulfation. In our previous study a significantly higher amount of free sialic acid was detected in blood serum from patients suffering of post-acute infectious syndromes (PAIS). It is known that neuraminidase-mediated cleavage of sialic acid from erythrocytes leads to increased lactate formation, cell shape alteration and enhanced mononuclear phagocytic clearance [1]. In our study we aim to evaluate erythrocyte glycocalyx damage leading to decreased surface charge, which can result in impaired microcirculation and O2-delivery in patients suffering from PAIS. Methods Erythrocytes from ME/CFS patients were collected and used in hemagglutination assays with sialic acid-specific lectin SNA (S. nigra). These were compared with healthy controls. N-glycan analysis of healthy erythrocyte membranes was performed. N-glycans were separated and relatively quantified using HPLC-FL and assigned based on MALDI mass spectrometry data. Rheology measurements were conducted of intact and neuraminidase-induced de-sialylated erythrocytes. Results Erythrocytes isolated from ME/CFS patients demonstrated significantly reduced hemagglutination compared to those from healthy controls. This finding is consistent with previous reports of elevated free sialic acid levels in serum and may indicate increased shedding of sialic acid from the erythrocyte surface. Analysis of surface-bound N-glycans revealed that di- and mono-sialylated glycan species were the predominant structures detected. Distinct rheological behaviors were observed between intact and desialylated erythrocytes. Specifically, in vitro neuraminidase treatment resulted in a marked reduction in erythrocyte flexibility, likely attributable to altered surface charge and membrane-associated properties. Conclusion The results point to a loss of sialic acid on erythrocytes from patients with disease. The resulting damage may cause a reduction of erythrocyte flexibility which negatively influences vascular capillary flow. N-glycan analysis of erythrocyte membranes from ME/CFS-affected individuals can be a useful method to accurately gauge the degree of surface sialic acid loss.