Gut issues are common with endurance exercise, ranging from mild discomfort to severe debilitating symptoms warranting reduced exercise workload or even cessation from activity. Considering the recent exponential growth of nighttime endurance and ultra-endurance events, athletes anecdotally report having more gut issues during and after night events compared to day events. To explore the impact of night exercise compared to day exercise on markers of gut integrity and function, systemic immune responses, gut symptoms and feeding tolerance, investigators recruited 16 trained endurance runners to undertake three hours of running on two separate occasions, from 0900h-1200h and 2100h-0000h. Investigators found that nighttime running results in greater disturbance to gut function, including slower gut transit, which translated to higher incidence and severity of gut symptoms. However, there were no differences in gut integrity, systemic immune markers and metabolic responses between the two running time periods. It was reported that the greater stress response (i.e., cortisol, but not catecholamines) seen in night running compared to day running likely promoted the disturbed gut function. This research highlights the need for endurance and ultra-endurance athletes undertaking nighttime events to assess pre- and during-exercise feeding strategies. It also highlights the need for future research to establish specific nighttime exercise feeding guidelines and recommendations.