This paper presents a study focusing on the influence of hysteretic heating on asphalt samples during laboratory fatigue testing. The experimental test setup for material characterization and temperature measurements, including its effect on fatigue test results, as well as theoretical aspects on hysteretic heating, are described. The experimental part of the investigation concerns linear viscoelastic and cyclic fatigue characterization of six asphalt concrete mixtures using uniaxial testing. All the mixtures show nominally identical volumetric properties (aggregate size distribution, binder and air void content) but different binder properties. Three base bitumens and three polymer modified binders were used. The cyclic fatigue tests were carried out at 0, 10, and 20°C using controlled strain and stress modes and different excitation amplitudes. In order to acquire knowledge regarding temperature changes during fatigue testing, several experimental techniques were used. The main thermal study was performed using thermocouples attached to the midheight envelope surface of each sample. The sample surface temperature distribution and its evolution during fatigue testing were investigated using an infrared thermal camera. Furthermore, a limited study of the magnitude of difference between surface and maximum temperature inside the sample was carried out using thermocouples embedded during gyratory compaction. When compared, each method shows advantages and disadvantages regarding simplicity and reliability. In principle, the three methods provide similar results, but the type of information obtained differs among the methods. The use of thermocouples attached to the envelope surface during fatigue testing provides accurate and consistent results of global temperature that can be used to investigate the influence of heating on asphalt fatigue characteristics. By use of thermal measurements and a continuum damage model, it was possible to show a pronounced effect of heating on fatigue behavior. The influence of heating was especially obvious at high excitation amplitudes and elevated temperatures, i.e., conditions where the material produces high amounts of viscoelastic dissipated energy as well as temperature sensitive material behavior.