Exercise physiology in Colombian Creole Horses remains poorly characterized despite the breed’s extensive use in athletic activities. The objective of this study was to evaluate exercise-induced changes in blood biochemical and gas parameters and to determine whether these responses differed according to sex, geographic origin, or gait modality. An analytical observational study with repeated measures was conducted in 76 athletic CCHs. Blood samples were obtained from the jugular vein at rest and immediately after exercise to measure acid–base indicators (pH, carbon dioxide pressure, bicarbonate), electrolytes (sodium, potassium, calcium, chloride), and metabolic variables (glucose, lactate, creatinine). Within-subject comparisons were performed to quantify exercise-induced changes, and subgroup analyses were conducted by sex, geographic region, and gait modality. Exercise resulted in physiologically consistent responses, including a mild reduction in pH (−0.01), decreases in carbon dioxide pressure (−2.39 mmHg) and bicarbonate (−1.7 mmol/L), and a moderate increase in lactate (+2.96 mmol/L), indicating exercise-associated metabolic acidosis and anaerobic metabolism. Glucose and creatinine also increased modestly, while electrolyte concentrations remained relatively stable. No meaningful differences were detected between sexes or geographic regions. As expected, gait modality significantly influenced acid–base responses, with horses performing trocha exhibiting greater reductions in carbon dioxide pressure and bicarbonate. These findings indicate that gait type modulates the magnitude of exercise-induced acid–base responses in Colombian Creole Horses.