A single endurance exercise session was shown to lead to a reduction of pain perception and an elevation of mood. We hypothesized that athletes, who regularly practice endurance, might also induce changes in mood and pain processing in expectation of an endurance session. We compared the expectation effects of a 2-h-run on mood and pain processing to a run-free control day (RFC). Fifteen trained runners were assessed with repeated painful and nonpainful pinprick stimulation in a functional magnetic resonance imaging (fMRI) scanner prior to a 2-h-run and at RFC. Pain ratings, pressure pain stimulus-response functions, and euphoria ratings were also assessed. There were no mean expectation effects on any of the behavioral measures. However, highly trained athletes needed more pressure to evoke a pain rating of 46 pre-run vs. RFC but were less euphoric pre-run vs. RFC. Furthermore, analysis of brain activities to painful stimuli applied immediately before the 2-h-run compared to RFC revealed increased activation in the medial prefrontal cortex (mPFC) and reduced activation in posterior insula. Additionally, less trained athletes had reduced activation in posterior insula to painful stimulation pre-run vs. RFC compared to highly trained athletes. The results suggest reduced central pain processing in expectation of an endurance run and an association between the amount of the expectation effect and training frequency which differs depending on the stimuli applied.