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Understanding changes to gut microbiota composition and metabolic output in response to acute exercise may be necessary for understanding the mechanisms mediating the long-term health and performance benefits of exercise. Our primary objective was to characterize acute changes in the fecal microbiome and metabolome following participation in an ultra-endurance (3.9 km swim, 180.2 km bike, 42.2 km run) triathlon. An exploratory aim was to determine associations between athlete-specific factors [race performance (i.e., completion time) and lifetime years of endurance training] with pre-race gut microbiota and metabolite profiles. Stool samples from 12 triathletes (9 males/3 females; 43 ± 14 yr, 23 ± 2 kg/m 2 ) were collected ≤48 h before and the first bowel movement following race completion. Intra- and inter-individual diversity of bacterial species and individual bacterial taxa were unaltered following race completion ( P > 0.05). However, significant reductions ( P < 0.05) in free and secondary bile acids [deoxycholic acid (DCA), 12-keto-lithocholic acid (12-ketoLCA)] and short-chain fatty acids (butyric and pivalic acids), and significant increases ( P < 0.05) in long-chain fatty acids (oleic and palmitoleic acids) were observed. Exploratory analyses revealed several associations between pre-race bacterial taxa and fecal metabolites with race performance and lifetime history of endurance training ( P < 0.05). These findings suggest that 1 ) acute ultra-endurance exercise shifts microbial metabolism independent of changes to community composition and 2 ) athlete performance level and training history relate to resting-state gut microbial ecology. NEW & NOTEWORTHY This is the first study to characterize acute changes in gut microbial ecology and metabolism following an ultra-endurance triathlon. We demonstrate changes in gut microbial community function, but not structure, as well as several associations between gut microbiome and fecal metabolome characteristics with race completion time and lifetime history of endurance training. These data add to a small but growing body of literature seeking to characterize the acute and chronic effects of exercise on the gut microbial ecosystem.