This study investigates the effect of periodic overloading on the fatigue life of Reinforced Concrete (RC) beams strengthened with externally bonded Fiber Reinforced Polymers (FRP). The study includes 6 RC beams with dimensions of 152.4 × 152.4 × 1500 mm strengthened with one layer of carbon fiber sheet attached to the soffits. One strengthened beam tested monotonically to serve as a control beam, and another one was tested under constant amplitude fatigue loading to serve as a reference for the fatigue testing. The rest of the beams were tested under fatigue loading with periodic overloading. The loading pattern was chosen to simulate the conditions of the traffic on bridges. Two sets of repetitive loading segments were applied with a ratio of (1:9), one overloading cycles to nine base loading cycles. When compared with the reference RC beam and the available data obtained from the literature; the results show that periodic overloading reduces the fatigue life of the strengthened beams. The Palmgren-Miner rule of linear cumulative damage overestimates the fatigue life of the strengthened beams. In addition, periodic overloading shifts the location of the failure in the strengthened beams outside the maximum moment zone. However, the tested beams had the same failure mode of the reference beam, which is the rupture of the primary reinforcing steel. In this study, a prediction model is presented of the fatigue life of strengthened RC beams subjected to overloading based on the applied stress ranges in the primary steel.