Abstract Background: This study investigates the variations of Heart Rate Variability (HRV) due to a real-life stressor and proposes a classifier based on nonlinear features of HRV for automatic stress detection. Methods: 42 students volunteered to participate to the study about HRV and stress. For each student, two recordings were performed: one during an on-going university examination, assumed as a real-life stressor, and one after holidays. Nonlinear analysis of HRV was performed by using Poincaré Plot, Approximate Entropy, Correlation dimension, Detrended Fluctuation Analysis, Recurrence Plot. For statistical comparison, we adopted the Wilcoxon Signed Rank test and for development of a classifier we adopted the Linear Discriminant Analysis (LDA). Results: Almost all HRV features measuring heart rate complexity were significantly decreased in the stress session. LDA generated a simple classifier based on the two Poincaré Plot parameters and Approximate Entropy, which enables stress detection with a total classification accuracy, a sensitivity and a specificity rate of 90%, 86%, and 95% respectively. Conclusions: The results of the current study suggest that nonlinear HRV analysis using short term ECG recording could be effective in automatically detecting real-life stress condition, such as a university examination