Dynamic Message Signs (DMSs) are increasingly used in highways as an effective means to communicate time-sensitive information with motorists. To ensure their long-term performance, it is critical to ensure that the truss structures that hold them can resist not only extreme loading events, but also fatigue induced by service loads. For this purpose, a comprehensive study has been conducted on the fatigue performance of this important category of structures. The current study evaluates the fatigue performance of DMS-support structures during transportation under road-induced excitations as well as throughout their service life under loads induced by environmental stressors. In the first section, fatigue analysis of these structures during transportation under road-induced excitations is conducted. To investigate this, a comprehensive field test and numerical study are conducted. For short-term monitoring, one span of a sign-support structure is instrumented. Additionally, detailed finite element simulations are conducted to obtain an in-depth understanding of the potential modes of damage under the road-induced excitations. The outcome of this study is expected to determine the extent of fatigue of DMS-support structures during transportation. In the second part, vulnerability assessment of these structures is investigated under thermal loads. For that purpose, two DMS-support structures located in Iowa are selected for long-term monitoring. Moreover, detailed finite element simulations are conducted to obtain an in-depth understanding of the expected extent of damage. Based on the results obtained from this study, the DMS-support structures are found to demonstrate a great performance under thermal loads, which strongly supports the recent transition from aluminum to steel truss structures. Finally, fatigue performance of DMS-support structures under the combined effects of diurnal temperature changes and natural wind excitations is investigated. Field monitoring has been paired with detailed FE simulations to understand the fatigue performance of DMS-support structures under multiple stressors. The current study is concluded with the investigation of wind directionality effects. The outcome of this study is expected to not only contribute to the long-term performance and safety of DMS-support structures, but also pave the way to implement similar multi-stressor perspectives for other transportation infrastructures in service.