The motion system is a major component of a driving simulator. It provides motion cues in various directions with different degrees of freedom (DOF). Different DOF could influence response variables widely used in highway design safety evaluation conducted in simulators. Few studies, though, have comprehensively explored the effect of driving simulator motion cues on driving performance. The high-fidelity Tongji University Driving Simulator (TUDS) was used to separately compare five dependent variables: speed, speed change, lane deviation, longitudinal acceleration, and lateral acceleration. The Wilcoxon signed-rank test was used to examine the difference for each variable using 8-DOF and fixed-base motion system configurations for nine types of combined highway alignments. A subjective assessment of participants’ driving experience was conducted, and the chi-square test for each term was used to compare the difference in motion system fidelity and adverse reactions between the two configurations. Results show that: (1) speed and lateral acceleration are more likely to be significantly affected by motion cues than speed change, lane deviation, and longitudinal acceleration; (2) the horizontal curve with up-grade is the type of combined alignment where speed and lateral acceleration are mostly affected; (3) characteristics of the five variables in different combined alignments vary between the two simulators; (4) the 8-DOF motion system provides better perceived motion fidelity than the fixed-base system does, and the 8-DOF simulator causes less discomfort and tiredness in simulator sickness; and (5) the presence of motion cues is suggested for highways with complex combined curves when focusing on speed and lateral acceleration.