OBJECTIVE: To investigate the effects of objectively measured habitual sleep patterns on cardiac autonomic modulation (CAM) in a population-based sample of adolescents. METHODS: We used data from 421 adolescents who completed the follow-up examination in the Penn State Children Cohort study. CAM was assessed by heart rate (HR) variability (HRV) analysis of beat-to-beat normal R-R intervals from a 39-h electrocardiogram, on a 30-min basis. The HRV indices included frequency domain (HF, LF, and LF/HF ratio), and time domain (SDNN, RMSSD, and heart rate or HR) variables. Actigraphy was used for seven consecutive nights to estimate nightly sleep duration and time in bed. The seven-night mean (SD) of sleep duration and sleep efficiency were used to represent sleep duration, duration variability, sleep efficiency, and efficiency variability, respectively. HF and LF were log-transformed for statistical analysis. Linear mixed-effect models were used to analyze the association between sleep patterns and CAM. RESULTS: After adjusting for major confounders, increased sleep duration variability and efficiency variability were significantly associated with lower HRV and higher HR during the 39-h, as well as separated by daytime and nighttime. For instance, a 1-h increase in sleep duration variability is associated with -0.14(0.04), -0.12(0.06), and -0.16(0.05) ms(2) decrease in total, daytime, and nighttime HF, respectively. No associations were found between sleep duration, or sleep efficiency and HRV. CONCLUSION: Higher habitual sleep duration variability and efficiency variability are associated with lower HRV and higher HR, suggesting that an irregular sleep pattern has an adverse impact on CAM, even in healthy adolescents.