This study aimed to analyze the impact of sleep deprivation (SD) on cardiac, hemodynamic, and endothelial parameters and to determine whether these are sustained with increased periods of SD. The study included 60 healthy men (mean: age 31.2±6.3 years; body mass index 24.6±2.6 kg/m2). Hemodynamic parameters, parameters of myocardial contractility, spectral analysis of heart rate (HR) and blood pressure (BP) variability, and the sensitivity of arterial baroreflex function were evaluated. Biochemical tests were performed to assess L-arginine (L-Arg) and asymmetric dimethylarginine (ADMA) levels in reflection of endothelial nitric oxide synthase ability. Measurements of cardiovascular system parameters were obtained at 9 a.m. (baseline) on the first day of the study and 9 a.m. (24-h SD), 1 p.m. (28-h SD), and 5 p.m. (32-h SD) on the second day. Blood samples for evaluating biochemical parameters were obtained at baseline and after 24-h SD. ANOVA Friedman's test revealed a significant effect for time in relation to HR (χ2=26.04, df=5, p=0.000), systolic BP (χ2=35.98, df=5, p=0.000), diastolic BP (χ2=18.01, df=5, p=0.003), and mean BP (χ2=28.32, df=5, p=0.000). L-Arg and ADMA levels changed from 78.2±12.9 and 0.3±0.1 at baseline to 68.8±10.2 and 0.4±0.1 after 24-hr SD, respectively (p=0.001, p=0.004). SD in healthy men is associated with increases in BP, which appear to occur after 24 hours of SD and are maintained over increasing periods of SD. The observed hemodynamic changes may have resulted due to disordered vascular endothelial function, as reflected in alterations in L-Arg and ADMA levels.