The random and varied longitudinal and transverse loading positions of vehicles exert different effects on the fatigue performances of bridges. Consequently, bridge components exhibit different structural failure mechanisms under heavy duty. To determine the effect of heavy duty on the fatigue performance of beams, this study conducted a fatigue test with load amplitude and loading method as variables. First, 10 test beams were designed, and the static test was performed on three of the beams. Second, fatigue amplitude was determined on the basis of static test results and actual vehicle data. Constant and variable amplitude loading tests were conducted on the seven remaining beams. Finally, the material properties of prestressed concrete beams under long-term heavy vehicular load were analyzed. Research results demonstrate that all test beams suffered from fatigue failure cause by brittle fracture that initiate in the bottom beam reinforcement. The strain variation of all test beams can be divided into three stages. The residual strain of steel bars is positively correlated with the number of loads. Under the fixed low-load limit, the fatigue life of test beams is shortened by 95% and 98%, respectively, when the upper load limit is 0.75 and 0.8 times of the ultimate load. The fatigue life of test beams under the low-high loading process is 46% shorter than that under the high-low loading process. The conclusions can provide theoretical references for the late performance evaluation and maintenance of prestressed concrete beams.