The sensitivity of physiological measures to mental workload was investigated in a flight simulator. Twelve pilots had to fly through a tunnel with varying levels of difficulty. Additionally, they had to perform a memory task with four levels of difficulty. The easiest memory task was combined with the easiest tunnel task and the most difficult memory task with the most difficult tunnel task. Between the tunnel tasks, subjects had to fly a pursuit task in which a target jet had to be followed. Rest periods before and after the experiment were used as a baseline for the physiological measures. Mental workload was measured with heart period, continuous blood pressure, respiration and eye blinks. Several respiratory parameters, heart rate variability, blood pressure variability and the gain between systolic blood pressure and heart period (modulus) were scored. All measures showed differences between rest and flight, and between the pursuit and the tunnel task. Only heart period was sensitive to difficulty levels in the tunnel task. Heart rate variability increased when respiratory activity around 0.10 Hz increased, which occurred often. The modulus was hardly influenced by respiration and therefore appears to be a better measure than heart rate variability. Among the respiratory parameters, the duration of a respiratory cycle was the most sensitive to changes in workload. The time in between two successive eye blinks (blink interval) increased and the blink duration decreased as more visual information had to be processed. Increasing the difficulty of the memory task led to a decrement in blink interval, probably caused by subvocal activity during rehearsal of target letters. The data show that physiological measures are sensitive to mental effort, whereas rating scales are sensitive to both mental effort and task difficulty.