A series of studies have suggested that stage N2 is vulnerable and strongly affected by the first-night effect (FNE). However, the neurophysiological mechanism underlying the vulnerability of stage N2 of the FNE has not been well examined. A total of 17 healthy adults (11 women and 6 men, mean age: 21.59 ± 2.12) underwent two nights of polysomnogram recordings in the sleep laboratory. We analyzed sleep structure and central and autonomic nervous system activity during stage N2 and applied the electroencephalographic (EEG) activation index (beta/delta power ratio) and heart rate variability to reflect changes in central and autonomic nervous system activity caused by the FNE. Correlation analyses were performed between EEG activation and heart rate variability. The results showed that EEG activation and high-frequency heart rate variability increased on the adaptation night (Night 1). Importantly, EEG activation was significantly associated with the percentage of stage N1, and the correlation between EEG activation and high-frequency heart rate variability decreased due to the FNE. These findings indicate that the FNE affects the instability of stage N2 by increasing central nervous system activity and uncoupling the activity between the central and autonomic nervous systems.