A number of studies have used heart rate variability (HRV) measures to estimate driver stress across different driving conditions. Understanding the constructs of driver stress can provide insights regarding the underlying reasons for safety-critical events. The intent of this study is to evaluate the electrocardiogram (ECG) activities of drivers along a pre-defined route with varying roadway conditions. Heart rate, standard deviation of selected RR interval series (SDNN) and low frequency to high frequency ratio (LF/HF) were examined. An examination of short interval stress at evenly distributed points along the route indicates that some impact was detected from long duration driving and the statistical model was adjusted accordingly to reflect this. The findings suggest that temporal and frequency domain parameters generated similar characteristics regardless of the direction of travel (clockwise or counterclockwise direction). That is, these two parameters provided fairly consistent outcomes regarding driver workload. However, heart rate was not as good an indicator suggesting that this measure may be picking up some other unexplained effects.