Real-life stressors, such as university examination, cause an increase in sympathetic activity of the nervous system innervating the heart, and thus an increase in heart rate (HR). Our study aimed to detect changes in heart rate variability (HRV) during different stages of an exam in a group of 90 healthy university students (30 males and 60 females), over 4 h of monitoring divided into 1 h before, 2 h during, and 1 h after the examination. HRV was significantly highest after the exam, indicating release from stress, as compared to before and during the examination when stress was observable. Undergraduate students in different academic years did not differ in terms of stress, indicating the absence of adaptation to exam procedures. However, HR and R-R interval after the exam showed significant difference between first year undergraduate studies and first year of a graduate program, indicating a higher degree of confidence in graduate students. Results also suggest that HRV in females is significantly lower than that in males before and after examination, despite men having greater sympathetic input. In conclusion, the results of our novel study assessing stress in real-time examination show important gender differences, and lack of adaptation with academic study year.