Gut-training has been shown to improve gastrointestinal tolerance, circulatory glucose availability, and exercise performance. The study aimed to investigate the effects of a repetitive feeding-challenge using fat versus carbohydrate (CHO) on markers of gastrointestinal function, glucose availability, and subsequent performance when challenged with a high-CHO load (87 g/hr) during exercise. Forty-four endurance athletes (mean ± SD [9 females and 35 males]: body mass: 71.2 ± 9.2 kg, height: 173.6 ± 7.0 cm, ${\dot{\mathrm{V}}\mathrm{O}}_2\text{max}$: 55.0 ± 6.1 ml·kg⁻¹·min⁻¹) completed a preintervention gut-challenge trial (T1), involving a 2 hr run (60% ${\dot{\mathrm{V}}\mathrm{O}}_2\text{max}$) while taking a CHO gel every 20 min (87 g/hr, 10% w/v), followed by a 1 hr self-paced distance test with ad libitum water. Participants were then randomized to a fat (fat feeding-challenge [FFC]; 20 g nut butter, 124 kcal, 11 g fat, 3 g protein, and 3 g CHO) or CHO supplement (CHO feeding-challenge [CFC]; 47 g CHO gel: 123 kcal, 29 g CHO) group to complete a 7-day repetitive feeding-challenge (1 hr exercise and supplement intake every 20 min with 290 ml water), followed by a gut-challenge retrial (T2). FFC did not differ from CFC in terms of resting orocecal transit time, feeding tolerance, or substrate oxidation during T1 and T2. Peak breath hydrogen was lower in FFC than CFC (p = .028) at T2. Total (FFC: 27%, p = .005 vs. CFC: 38%, p = .001) and upper gastrointestinal symptoms severity (FFC: 26%, p = .013 vs. CFC: 40%, p < .001) during exercise was reduced similarly between groups from T1 to T2. FFC covered more distance in T2 (11.51 ± 2.02 vs. 11.08 ± 2.02 km, p = .013), but not significantly different to CFC (p = .341). A repetitive feeding-challenge with fat does not enhance nor worsen gastrointestinal and fueling outcomes compared with a CHO repetitive feeding-challenge.