A suspension unit is a significant component in a vehicle. It provides a smooth ride, improves traction, maintains ride geometry and facilitates better handling. A typical suspension setup in a two-wheeler consists of a front and rear suspension setup working independently to achieve the desired function. It connects the wheel to the chassis of the vehicle, transferring road load and hence affecting the durability of the structural members. This research work focuses on the durability aspect of the suspension unit with its preload as the control parameter. A rear suspension with seven preload settings was chosen for this study. The provision requirements of preload settings in suspension were experimentally analyzed using static measurements. The vehicle was instrumented with transducers (strain gauges, accelerometers, linear displacement sensors, and Global Positioning System (GPS) sensors), and dynamic road load data was acquired at different rear suspension preload settings by changing from the minimum, i.e., the 1st setting, to the maximum, i.e., the 7th setting, while keeping other parameters constant. Statistical analysis and fatigue analysis were utilized to study the dynamic effects. It was observed and quantified that increasing the preload for higher static load conditions or rough road conditions provides a smoother ride and improved life due to lower bump stopper engagement and full bump conditions owing to increased compression stroke availability.