Fatigue has major implications for transportation system safety; therefore, investigating the psychophysiological links to fatigue could enhance our understanding and management of fatigue in the transport industry. This study examined the psychophysiological changes that occurred during a driver simulator task in 35 randomly selected subjects. Results showed that significant electroencephalographic changes occur during fatigue. Delta and theta activity were found to increase significantly during fatigue. Heart rate was significantly lower after the driving task. Blink rate also changed during the fatigue task. Increased trait anxiety, tension–anxiety, fatigue–inertia and reduced vigor–activity were shown to be associated with neurophysiological indicators of fatigue such as increased delta and theta activity. The results are discussed in light of directions for future studies and for the development of a fatigue countermeasure device.