Debilitating gastrointestinal symptoms is a common feature of endurance running and may be exacerbated by and/or limit the ability to tolerate carbohydrate intake during exercise. The study aimed to determine whether two weeks of repetitive gut-challenge during running can reduce exercise-associated gastrointestinal symptoms and carbohydrate malabsorption. Endurance runners (n=18) performed an initial gut-challenge trial (GC1) comprising 2-hour running exercise at 60% VO2max (steady state) while consuming a formulated gel-disk containing 30g carbohydrates (2:1 glucose-fructose, 10% w/v) every 20minutes, followed by a 1-hour running effort bout. Gastrointestinal symptoms, feeding tolerance, and breath hydrogen (H-2) were determined along the gut-challenge trial. After GC1, participants were randomly assigned to a blinded carbohydrate (CHO, 90gCHOhour(-1)) or placebo (PLA, 0gCHOhour(-1)) gut-training group. This comprised of consuming the group-specific feeding intervention during 1-hour running exercise at 60% VO2max equivalent, daily over a period of two weeks. Participants then repeated the gut-challenge trial (GC2). In GC2, a reduced gut discomfort (P=.012), total (P=.009), upper- (P=.015), and lower-gastrointestinal (P=.008) symptoms, and nausea (P=.05) were observed on CHO, but not PLA. Feeding tolerance did not differ between GC1 and GC2 on CHO and PLA. H-2 peak was attenuated in GC2 (6 +/- 3ppm) compared to GC1 (13 +/- 6ppm) on CHO (P=.004), but not on PLA (GC1 11 +/- 7ppm, and GC2 10 +/- 10ppm). The effort bout distance was greater in GC2 (12.3 +/- 1.3km) compared with GC1 (11.7 +/- 1.5km) on CHO (P=.035) only. Two weeks of repetitive gut-challenge improve gastrointestinal symptoms and reduce carbohydrate malabsorption during endurance running, which may have performance implications.