The aim of this study was to (1) examine the presence of stress on professors when they teach in front of 200 students and analyse objectively such stress using biomarkers such as salivary cortisol, chromogranin A (CgA) and alpha-amylase (AA) (2) investigate whether sex affects the reactivity of salivary alpha-amylase (sAA) and cortisol concentrations and the interaction of both hormonal systems. Fifty-two participants (26 women and 26 men) collected nine unstimulated saliva samples on 2 days, (one working day, and one resting day). Cortisol concentrations and AA activity measured on the teaching day were significantly higher than those noted on the resting values. No differences between the resting day and the teaching day were reported for CgA. Our results showed a cortisol response to teaching, which was characterized by an anticipatory rise. The AA level was significantly increased after the end of the lecture, and returned to the pre-lecture level 30 min after the end of the lecture. The awakening cortisol response noted on the teaching day was significantly higher in females than in males. No baseline sex differences in sAA and CgA were observed and men and women seem to have a comparable reactivity in sAA, CgA and cortisol levels on lecture stress. The mechanisms that leads to modify activity of salivary AA and CgA due to stress is not entirely understood and further research is needed to elucidate them.