In horses, both the post exercise distribution of lactate between plasma and red blood cells (RBC) and the activity of lactate transporters on the RBC membrane vary widely between individuals. In this study, we investigated the effects of pH, time and temperature on lactate distribution in vitro, and compared the in vitro activity of lactate transporters with the accumulation of lactate into RBC in vivo. To accomplish this, we took venous blood samples at rest and after trotting races. The post exercise accumulation of lactate into RBC was shown to depend on the activity of lactate transporters. The results, in vitro, also indicate that pH, incubation time and temperature influence the activity of lactate transporters and the accumulation of lactate into RBC, underscoring the fact that in practice it is important to standardise the measurement conditions of lactate. These results support the view that whole blood lactate concentrations should be measured in estimating the accumulation of lactate from exercising muscles into the blood, because the effect of blood pH, temperature, time to centrifugation of the sample and also interindividual variation in lactate transport into RBC are therefore minimised.