BACKGROUND: Despite the importance of the gut microbiome on physical performance and health, little is known on the impact of training on an athlete's gut health. OBJECTIVE: This study investigates the effect of training load on markers of gut health. METHODS: Whole stool (24 h) samples were collected from 23 highly trained rowers (mean ± SD; age 19.2 ± 1.1 y; weight 80.1 ± 11.4 kg; height 1.83 ± 0.09 m) following periods of high (HT) and low training load (LT). The microbiome and short-chain fatty acid concentrations were characterized from the whole stool samples. Three-day weighted food records were used to determine diet quality (ADIcore), macronutrient, and fiber intakes during HT and LT. RESULTS: By design, training duration (147%) and intensity (130%) were greater during (HT), compared with (LT) ( < 0.001). Carbohydrate, fat, protein, and fiber intake remained stable, but ADIcore was higher in HT (55 ± 10) compared with LT (49 ± 9; t(15) = 2.78,  0.014; CI: 1.34 to 10.155). Stool frequency (1.11 ± 0.47 vs 0.67 ± 0.76;  0.007) was lower in HT compared with LT, and a greater number of participants were unable to produce a stool sample during LT (8% vs 47%). Short chain fatty acid (SCFA), propionic (120.64 ± 30.06 mm vs 91.35 ± 34.91 mm;  0.007), and butyric acid (104.76 ± 50.02 vs 64.23 ± 22.05 mm,  0.003) concentrations were lower in HT compared with LT. Alpha diversity, Shannon-Wiener diversity index (3.43 ± 0.37 vs 3.67 ± 0.34,  0.09) was lower in HT than LT. The abundance of the dominant was greater at HT compared to LT and ratio of firmicutes to ( = 16, 1.31 ± 1.19 vs 4.29 ± 3.88, t(15) = -3.44,  0.04, CI = -4.82 to -1.13) was lower in HT compared to LT. CONCLUSION: Results of this study indicate that gut microbiome, SCFA concentrations, stool frequency, and diet quality vary between periods of high and low training load in athletes. The relationship between these factors and impact of such changes in gut health is currently unclear and warrants further investigation.