Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar–capillary barrier. To determine the extent of the fluid exchange across the alveolar–capillary barrier at rest and during exercise, six horses were exercised on a high‐speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (ΔBV; measured in percentage) were calculated from changes in plasma protein and haemoglobin concentration, and haematocrit. Cardiac output (
Q) was calculated using the Fick equation. Fluid flux ( JV−A; measured in l min−1) across the alveolar–capillary barrier was then quantified based on Qand ΔBV. At rest, no fluid movement occurred across the pulmonary vasculature (0.6 ± 0.6 l min−1). During exercise, the amount of fluid moved from the pulmonary circulation was 8.3 ± 1.3 l min−1 at 1 min, 6.4 ± 2.9 l min−1 at 2 min, 10.1 ± 1.0 l min−1 at 3 min, 12.9 ± 2.5 l min−1 at 4 and 9.6 ± 1.5 l min−1 at fatigue (all P< 0.0001). Erythrocyte volume decreased by 6% ( P< 0.01) across the lungs, which decreased the colloid osmotic gradient in the pulmonary vasculature. Decrease colloid osmotic gradient along with increased hydrostatic forces in the pulmonary vasculature would enhance displacement of fluid into the pulmonary interstitium. In conclusion, exercise caused large increases in transpulmonary fluid fluxes in horses. Here, we present a simple method to calculate transpulmonary fluid fluxes in different species, which can be used to elucidate mechanisms of lung fluid balance in vivo.