The effect of lactate minimum speed (LMS)-guided training on horses’ homeostasis is still unknown. Thus, this study aimed to evaluate the effect of an LMS-guided training program on the fluid, electrolyte and acid-base status of horses. Ten untrained Arabian horses were submitted to an LMS test on a treadmill before and after six weeks of training. The training intensity was 80% of the LMS in the first three sessions and 100% of the LMS in the other sessions. The venous blood was collected before (T-1) and after (T-2) training at rest, during and after the LMS test for lactate, pH, pCO₂, HCO₃⁻, and electrolyte measurements. The LMS and strong ion difference (SID₄) were calculated. A mild increase in the mean values (p > 0.05) was observed at rest in T-2 in comparison with T-1 in the following variables: pH (from 7.436 ± 0.013 to 7.460 ± 0.012), pCO₂ (from 42.95 ± 1.58 to 45.06 ± 0.81 mmHg), HCO₃⁻ (from 27.01 ± 1.02 to 28.91 ± 0.86 mmol/L), and SID₄ (from 33.42 ± 1.45 to 35.06 ± 2.94 mmol/L). During T-2, these variables were more stable than during T-1. Despite the improvement in fitness, the LMS did not indicate a significant difference (from 5.40 ± 0.55 to 5.52 ± 0.20 m/s). The results confirmed that the LMS-guided training program had a positive impact on the horses’ acid-base status, although some adaptations are still required to improve their fitness.

Physical activity is recognized as a modulator of the intestinal microbial metabolism in humans and may also influence the microbiota of horses subjected to physical conditioning. While the physiological benefits of submaximal training are well-documented, its effects on the intestinal microbiota of horses that are fed a forage-only diet remain insufficiently understood. This study evaluated the fecal microbial composition of pasture-kept horses at three timepoints: before training (Submax), after seven weeks of exercise (Week7), and after 30 days of recovery (Trev30). Compositional analysis of the microbiota was explored by sequencing the V4 region of the 16S rRNA gene. A stable forage-only diet, confirmed by bromatological analysis, indicates the observed microbial shifts were primarily exercise-induced. Significant temporal dynamics in beta diversity (p = 0.001) indicated a marked shift in microbial community structure after training (Week7), with a subsequent partial restoration after recovery (Trev30). No significant changes in alpha diversity were detected (p > 0.05). A transient increase in the phylum Proteobacteria at Week7, which decreased by Trev30, suggested microbial adaptation to exercise-induced metabolic demands. LEfSe analysis identified the genera Rummeliibacillus (trained) and Hungateiclostridiaceae (controls) as discriminant between groups. Fecal pH showed a slight reduction after seven weeks, without statistical differences, and returned to equilibrium values in Trev30. Submaximal training induced a reversible modulation of the equine gut microbiota, demonstrating its adaptive capacity while maintaining overall ecological stability.