Long duration of driving is a significant cause of fatigue-related accidents on motorways or major roadways. The fatigue caused by driving for extended periods acutely impairs driver alertness and performance and can compromise transportation safety. This study quantitatively measured the progression of driver fatigue and identified the conservative safe duration of continuous highway driving. Thirty young male subjects were analyzed during 90 min of laboratory-simulated highway driving. Sleepiness ratings (SSS) and reaction time (RT) tests were used to assess impairment of driver alertness and vigilance. Additionally, various measures of driving performance recorded throughout the experiment were used to measure temporal deterioration of driver performance from alert to fatigued using principal component analysis (PCA). The analytical results revealed that SSS scores, reaction times (RTs) and unstable driving performance significantly increased over time, indicating that excessive driving time is a significant fatigue factor and potential cause of fatigue-related accidents. Moreover, the analytical results indicated that 80 min was the safe limit for monotonous highway driving. Based on the experimental findings of this study, public awareness of the adverse affects of driver fatigue during long-distance driving should be enhanced. This study provides explicit information of fatigue development that can be used to prevent fatigue-related accidents.