The novel aim of this study was to describe the reference values of different haematological and biochemical parameters in the Spanish purebred horse (Andalusian, SPB) in each of the stages of a programmed exercise on a treadmill system, and to establish heritability and genetic correlations for these haematological and biochemical parameters. For this, 94 young SPB male horses (4.22 ± 2.27 years old) were used. An increasing intensity exercise test at 4, 5, 6 and 7 m/s was carried out on a treadmill (6% inclination). Total red blood cells, total white blood cells, neutrophils and lymphocytes counts; haematocrit, haemoglobin, lactate, uric acid, creatinine and total plasma proteins concentrations and aspartate transaminase, lactate dehydrogenase, creatine-quinase activities were determined. To conclude: (i) the reference values for each parameter were determined for each of the exercise test stages (ii) all the parameters analysed manifested a medium-high heritability and a high repeatability. These results will, in the near future, determine the measuring guidelines for improving the SPB horse's athletic ability on an objective treadmill system and for selecting these animals in response to those parameters.