STUDY OBJECTIVES: Short sleep duration and poor sleep quality are associated with adverse cardiovascular outcomes. Potential pathophysiological mechanisms include sleep-associated alterations in the autonomic nervous system. The objective of this study was to examine the associations of shorter sleep duration and poorer sleep quality with markers of autonomic tone: heart rate (HR), high-frequency HR variability (HF-HRV) and salivary amylase. METHODS: Cross-sectional analysis of data from actigraphy-based measures of sleep duration and efficiency and responses to a challenge protocol obtained from 527 adult participants in the Multi-Ethnic Study of Atherosclerosis. RESULTS: Participants who slept fewer than 6 h per night (compared to those who slept 7 h or more per night) had higher baseline HR (fully adjusted model 0.05 log beats/min, 95% confidence interval [CI] 0.01, 0.09) and greater HR orthostatic reactivity (fully adjusted model 0.02 log beats/min, 95% CI 0.002, 0.023). Participants who slept 6 to less than 7 h/night (compared to those who slept 7 h or more per night) had lower baseline HF-HRV (fully adjusted model -0.31 log msec(2), 95% CI -0.60, -0.14). Participants with low sleep efficiency had lower baseline HF-HRV than those with higher sleep efficiency (fully adjusted model -0.59 log msec(2), 95% CI -1.03, -0.15). Participants with low sleep efficiency had higher baseline levels of amylase than those with higher sleep efficiency (fully adjusted model 0.45 log U/mL, 95% CI 0.04, 0.86). CONCLUSIONS: Short sleep duration, low sleep efficiency, and insomnia combined with short sleep duration were associated with markers of autonomic tone that indicate lower levels of cardiac parasympathetic (vagal) tone and/or higher levels of sympathetic tone.