The entrance and exit sections of a tunnel are the accident black-spots in an expressway. For a safe operation of road tunnels, it is necessary to understand a driver’s physiological indices and driving performance when driving through tunnels. In this study, the UC-Win/Road simulation software was used to build 12 tunnel models of different lengths. A simulated driving experiment was carried out in a 6-DoF motion platform. The lateral position of vehicles characterizing the driving performance was measured using the motion platform. Electrocardiogram and eye movement data of 25 recruited drivers were collected simultaneously through the experiment. The spatial changes in a driver’s heart rate (HR) growth rate, RMSSD, pupil diameter growth rate and vehicle lateral deviation within 300 m before and after the tunnel entrance and exit were analyzed to determine the variation rules in the different tunnels. The study identified the length range in the tunnel entrance and exit sections that influences the drivers. A quantitative analysis was further carried out to analyze the relationship between the physiological indices and the driving performance indicator. The results showed that a driver’s heart rate fluctuates significantly 250 m before the tunnel entrance and 50 m before the exit. In this region, the pupil diameter increases gradually, and drivers tend to shift the vehicle to the left. At the tunnel exit, the HR and RMSSD are affected significantly by the tunnel length, and the variation is higher in longer tunnels. In comparison, the tunnel length has no significant effect on the physiological indicators and driving performance of the drivers at the entrance and exit.