It is well known that highway bridges are subjected to fatigue as they work under live loads with different frequencies and amplitude. The safety level for fatigue required by Brazilian codes is still unknown, especially for prestressed concrete girders. Also, current studies on fatigue reliability of bridges only evaluate bending. This work assesses the fatigue safety level provided by Brazilian design standards for a concrete highway bridge, using weigh-in-motion (WIM) data of an important federal Brazilian highway, BR-381 (Fernão Dias Highway). The Palmgren-Miner rule is considered to evaluate the service life and reliability indexes, from the fatigue point of view, of prestressed girders designed according to Brazilian codes. Using limited and complete prestressing levels, different traffic volumes are considered. It is found that the fatigue safety levels of longitudinal and transverse reinforcements are larger than the ones recommended by the international literature.