This paper investigates the effects of shiphandling manoeuvres on mental workload and physiological reactions in ten marine pilots. Each pilot performed four berthings in a ship simulator. Those berthings were differentiated by two factors, level of difficulty and familiarity with the port. Each berthing could also be divided into five phases, three during the execution and two resting periods, one before and one after the execution (dedicated to baseline physiological data collection). Mental workload was measured through two self assessment scales: the NASA TLX and a Likert scale. Power spectral densities on Beta bands 1 and 2 were obtained from EEG. Heart rate and heart rate variability were obtained from ECG. Pupil dilation was obtained from eye tracking. Workload levels were higher as berthings increased in difficulty level and/or the pilots completed the berthings in unfamiliar ports. Responses differed across specific phases of the berthings. Physiological responses could indirectly monitor levels of mental workload, and could be adopted in future applications to evaluate training improvements and performance. This study provides an example of an applied methodology aiming to define an upper redline of task demands in the context of marine pilotage.