Many researchers have proposed that challenging behaviors emitted by individuals with Autism Spectrum Disorder are related to abnormal physiological arousal. It has been suggested that behaviors such as stereotypy and self-injury function to regulate arousal and to reduce the discomfort associated with hypo- or hyper-arousal. Little empirical research has tested these theories. The current study investigated heart rate during challenging behavior in three children diagnosed with Autism Spectrum Disorder. Heart rate before, during, and after challenging behaviors was analysed. Specific heart rate patterns were found to co-occur with challenging behaviors. Abnormal heart rate responses to stressors were also noted. These findings offer little support for the arousal modulation theories of challenging behavior. We suggest that, for some individuals with Autism Spectrum Disorder, these behaviors serve to increase arousal and to allow, or sustain, access to a preferred state of heightened arousal. These findings, which are not wholly in line with previous research, may have implications for the assessment and treatment of challenging behavior.