Emotion regulation may influence psychological responses to exercise. We examined whether the emotion regulation strategies, cognitive reappraisal and distraction, influenced psychological state and prefrontal cortex oxygenation during endurance exercise. Twenty-four endurance runners ran for 90 min at 75–85% maximum heart rate in three separate sessions with no instruction or with instructions to use cognitive reappraisal or distraction. Participants rated their emotional arousal, emotional valence, and perceived exertion before, every 30 min during, and after exercise. Functional near-infrared spectroscopy quantified changes in prefrontal cortex oxygenation. Participants felt lower emotional arousal and physical exertion when instructed to utilize cognitive reappraisal than when given no emotion regulation instruction. Such responses to distraction did not differ from the other conditions. Emotion regulation strategies did not influence emotional valence or prefrontal cortex oxygenation. Participants’ analytical interpretation of the cognitive reappraisal instruction could contribute to small effect sizes and limited effects. Further research should determine contexts under which emotion regulation strategies most benefit endurance exercise experience.