Study objectives: This case-control study aimed to identify and validate behavioral markers supporting the diagnosis of disorders of arousal (DOA) with video polysomnography. Methods: All behaviors associated with 1,335 episodes of N3 interruptions were compared in 52 adult patients with DOA versus 52 participants without DOA (healthy control patients and patients with insomnia, hypersomnia, or sleep apnea syndrome). Results: Patients with DOA had more frequent (5.1 ± 2.4 versus 3.4 ± 1.9 interruptions/N3 time) and longer (35.8 ± 33 versus 23.1 ± 21.4 sec) arousals and awakenings from N3 than control patients. In the DOA group, the onset of behaviors was more abrupt, and behaviors including eye opening (69% versus 16%), head raising (41% versus 9%), visually exploring the environment (27% versus 1%), expression of fear/surprise (21% versus zero), speaking (18% versus 0.3%), trunk raising (13% versus 0.3%), and interacting with the environment (13% versus 0.5%), were (unlike quiet, comfort behaviors) more frequent than in control patients. A cutoff of two or more N3 interruptions containing eye opening yielded a sensitivity of 94.2% and a specificity of 76.9% for a DOA diagnosis. This accuracy was confirmed in a second set of data (second night of monitoring). Behaviors including an expression of fear/surprise (67.3%), sitting (32.7%), screaming, and standing up were specific to patients with DOA. Conclusions: A simple, behavioral video marker of behavioral reactions during N3 interruption (ie, opening the eyes at least two times in the same night) was sensitive, specific, and reproducible for discriminating patients with DOA from sleep laboratory control patients.