Manipulation of the mouse gut microbiome has been shown to increase gut-derived short-chain fatty acids and improve exercise capacity. Associations between exercise performance and gut microbiome composition and metabolites have also been identified in human studies. Yet there is little direct evidence that prebiotics are able to increase acetate production and improve exercise capacity in human participants. We conducted a randomised controlled cross-over trial with 21 healthy and active males (35.0 ± 6.9 years; 24.4 ± 2.7 kg/m2) to investigate the effect of 15 g of inulin (prebiotic) on exercise performance (15 km cycle time trial), compared to placebo. Time to completion of a 15 km time trial was the primary outcome, while plasma acetate concentration and markers of inulin fermentation (breath H2 concentration) and muscle oxygenation were measured to explore potential mechanisms of action. Time to complete the 15 km time trial was not affected by inulin mean difference between inulin and placebo trials: (−10.37 s, 95% CI [−150.8, 130.1 s], p = 0.884). The marker of inulin fermentation (H2 concentration increase from baseline) was significantly higher in inulin compared to placebo condition (+42.61 ppm, 95% CI [30.04, 55.19], p = 0.001 and +31.13 ppm, 95% CI [3.73, 58.51], p = 0.029, respectively), but plasma acetate concentration did not differ between conditions. Likewise, markers of muscle oxygenation were not different between inulin and placebo. Our current results do not support the acute use of prebiotics to improve exercise performance in adults. Possible explanations for the absence of ergogenic effects may be that the timing between prebiotic ingestion and exercise was too short to allow for complete fermentation into acetate, participants were in a fasted rather than a fed state, or that the single dose of supplement was insufficient. These factors, together with advanced methods (stable isotope studies) should be investigated in a follow-up study to elucidate the fate and role of colonic-derived acetate during exercise.