A growing body of research has suggested heart rate variability (HRV) may be reduced in autism spectrum disorder (ASD) in comparison to neurotypical cohorts. While there have been several studies investigating HRV in children diagnosed with ASD, few studies have been conducted in adults. The objective of the current study was to investigate autonomic nervous system activity as assessed by HRV in adults diagnosed with ASD. We hypothesized that adults with ASD would show a reduction in HRV compared to neurotypical participants. Participants diagnosed with ASD (n = 55) were recruited from the Autism Clinic for Translational Research at the Brain and Mind Centre (University of Sydney) between 2013 and 2017. Neurotypical participants were recruited from advertisements and online media. Resting state heart rate was measured for 5 min while participants sat in an upright position. Results showed there was an overall significant difference in resting-state HRV between adults diagnosed with ASD compared to the neurotypical control group. Logarithmically transformed high frequency (HF) and root mean square of successive differences were particularly decreased in the ASD group, suggesting lower parasympathetic activity. The use of psychotropic medications and comorbidities were associated with reductions in low frequency of HRV. Our data suggest an overall dysregulation in resting autonomic activity in adults with ASD. This may represent an important physiological mechanism leading to potential cardiovascular risk in ASD, which warrants further investigation. Autism Res 2019, 12: 922–930.