Vasovagal syncope (VVS) is the most common cause of syncope. Some stages of its pathophysiological mechanisms remain unclear. Vasoactive substances such as nitric oxide metabolites (NOx) and endothelin (ET) may be involved during acute orthostatic stress. To analyze plasma changes in NOx and ET and heart rate variability (HRV) in the supine positions (T1) and during the head-up tilt test (HUTT) (T2), in patients with VVS (case group) and control group. Thirty-seven patients (17 in the case group and 20 in the control group), matched for age and sex (mean aged 31.8 years) underwent HUTT with simultaneous HRV recording and venipuncture. Blood samples were collected during phases T1 and T2 and the analysis was performed without knowledge of the HUTT result. In the total sample, there was an increase in NOx values (P = .014), however there was no increase in ET values from phase T1 to phase T2. Patients with VVS tended to increase plasma NOx values (P = .057) and had significantly higher plasma values compared to ET (P = .033) between phases T1 to T2. In the control group, there was no significant change in the values of these vasoactive substances. Regarding HRV, there were a decrease in the component HF (high frequency) and increased of the LF (low frequency)/HF ratio during HUTT. There was an increase in ET during HUTT occurred only in the case group. These patients are more likely to have an imbalance between antagonistic vasoactive biomarkers during orthostatic stress.