Recent studies performed in humans and rats have reported that exercise can alter the intestinal microbiota. Athletic horses perform intense exercise regularly, but studies characterizing horse microbiome during aerobic conditioning programs are still limited. Evidence has indicated that this microbial community is involved in the metabolic homeostasis of the host. Research on ergogenic substances using new sequencing technologies have been limited to the intestinal microbiota and there is a considerable demand for scientific studies that verify the effectiveness of these supplements in horses. L-carnitine and chromium are potentially ergogenic substances for athletic humans and horses since they are possibly able to modify the metabolism of carbohydrates and lipids. This study aimed to assess the impact of acute exercise and aerobic conditioning, associated either with L-carnitine or chromium supplementation, on the intestinal microbiota of fillies. Twelve "Mangalarga Marchador" fillies in the incipient fitness stage were distributed into four groups: control (no exercise), exercise, L-carnitine (10g/day) and chelated chromium (10mg/day). In order to investigate the impact of acute exercise or aerobic conditioning on fecal microbiota all fillies undergoing the conditioning program were analyzed as a separate treatment. The fillies underwent two incremental exercise tests before and after training on a treadmill for 42 days at 70-80% of the lactate threshold intensity. Fecal samples were obtained before and 48 h after acute exercise (incremental exercise test). Bacterial populations were characterized by sequencing the V4 region of the 16S rRNA gene using the MiSeq Illumina platform, and 5,224,389 sequences were obtained from 48 samples. The results showed that, overall, the two most abundant phyla were Firmicutes (50.22%) followed by Verrucomicrobia (15.13%). The taxa with the highest relative abundances were unclassified Clostridiales (17.06%) and "5 genus incertae sedis" from the phylum Verrucomicrobia (12.98%). There was a decrease in the phylum Chlamydiae and in the genus Mycobacterium after the second incremental exercise test. Intense exercise changed the community's structure and aerobic conditioning was associated with changes in the composition and structure of the intestinal bacterial population of fillies. The intra-group comparison showed that chromium or L-carnitine induced moderate changes in the fecal microbiota of fillies, but the microbiota did not differ from the control group, which was exercised with no supplementation. Fecal pH correlated positively with Simpson's index, while plasma pH correlated negatively. Our results show that exercise and aerobic conditioning can change in the microbiota and provide a basis for further studies enrolling a larger number of horses at different fitness levels to better understand the effects of exercise and training on the intestinal microbiota of horses.