Acute lymphoblastic leukemia (ALL) and its treatment are associated with endothelial dysfunction (ED) and increased cardiovascular risk in adulthood. There are no data on ED in children after successful treatment of ALL. We aimed to assess new ED in these children using the plethysmographic reactive hyperemia index (RHI) and biomarkers that are known to be related to ED. In all, 22 children (mean 15.6 years), after successful treatment of ALL, and 18 healthy subjects were included in this prospective study. RHI, plasma concentrations of asymmetric dimethyl arginine (ADMA), high-sensitive CRP (hsCRP) and E-selectin were measured in all children. RHI values were significantly lower in ALL patients when compared with healthy controls (p<0.05). hsCRP was significantly increased in ALL patients compared with the control group (p<0.001). E-selectin plasma levels were higher in ALL patients as compared to healthy controls (p=0.05). This is the first study that combines both plethysmographic and biochemical methods to assess ED in ALL survivors. Significantly decreased RHI with elevated plasma concentrations of biochemical markers imply a possible association with premature ED in ALL patients. The combined diagnostic approach seems to be a valuable tool for more accurate detection of ED and preventive cardiovascular management in these patients.