Emotional stability plays a key role in individual and team performance during both routine activities and management of unexpected emergencies. Using a psycho-physiological approach, the stress response was investigated in drone operators in service.Methods: Salivary α-amylase (sAA), galvanic skin response (GSR) and anxiety were assessed over a 2-h operating flight.Results: Compared to baseline values, GSR and sAA values increased in operating conditions. Moreover, these values were higher in Pilots than in Sensor Operators, indicating that their stress response was greater. These results were associated with an increase in anxiety level, highlighting a relationship between autonomic reactivity and anxiety.Conclusion: This is the first report providing experimental evidences of the stress response related to Remotely Piloted Aircraft operations.