Background: The current study was conducted to evaluate the relation of sleep duration and quality with blood pressure (BP) and heart rate variability (HRV). Materials and Methods: This cross-sectional study was carried out in 2017 among 260 staff of a university hospital in Isfahan, Iran. They were selected by multi-stage random method from different wards. Time domain spectral analysis was used to measure a number of HRV parameters. The long-term components of the HRV were estimated using the standard deviation of the normal-to-normal interval (SDNN). The square root of the mean squared differences of successive NN intervals (RMSSD) was calculated by statistical time domain measurements; SNN50, and PNN50 were measured. Pittsburg sleep quality index (PSQI) questionnaire was used to assess sleep quality. Results: Higher PSQI score correlated with lower SDANN rise (OR=0.92). Fairly bad to very good subjective sleep quality had association with lower SDANN (OR=0.43). Very high sleep latency to very low sleep latency ratio had association with lower SDANN (OR=0.39) and lower PNN50 (OR= 0.44). Sleep duration and HRV parameters had no significant association. Fairly bad sleep efficiency to very good sleep efficiency ratio was correlated with lower SDANN (OR= 0.29). Very high daytime dysfunction to very low daytime dysfunction ratio had correlation with lower SDANN (OR=0.35). Very bad compared to very good subjective sleep quality had significant correlation with higher Heart rate (HR) (B=0.03). Very high sleep latency compared to no sleep latency was associated with higher HR (B=4.74). Very high compared to very low amount of sleep disturbances correlated with higher SBP levels (B=15.2). Using sleep medication less than once a week compared with no history of taking such drugs was associated with higher HR (B=16.4). Conclusion: Our findings showed that poor sleep quality are adversely associated with HRV, HR and BP. This finding should be considered in clinical and preventive recommendations.