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 vs. 52 participants without DOA (healthy controls and patients with insomnia, hypersomnia or sleep apnea syndrome). Results: Patients with DOA had more frequent (5.1 ± 2.4 vs. 3.4 ± 1.9 interruptions/N3 time) and longer (35.8 ± 33 vs. 23.1 ± 21.4 sec) arousals and awakenings from N3 than controls. In the DOA group, the onset of behaviors was more abrupt, and behaviors including eye opening (69 vs. 16%), head raising (41 vs. 9%), visually exploring the environment (27 vs. 1%), expression of fear/surprise (21 vs. 0%), speaking (18 vs. 0.3%), trunk raising (13 vs. 0.3%), and interacting with the environment (13 vs. 0.5%), were (unlike quiet, comfort behaviors) more frequent than in controls. A cutoff of 2 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. Conclusion: A simple, behavioral video marker of behavioral reactions during N3 interruption (i.e., opening the eyes at least two times in the same night) was sensitive, specific and reproducible for discriminating patients with DOA from sleep laboratory controls.