The aim of the present study was to investigate the effect of pentastarch on colloid osmotic pressure (COP) and cardiopulmonary function during and up to 24 h after anaesthesia in horses. Twenty-five systemically healthy horses were anaesthetised using isoflurane-medetomidine balanced anaesthesia. Twelve were assigned to treatment with hydroxyethyl starch (HES) (H group) and 13 to no HES (NH group). In the H group, 6 mL/kg of pentastarch 10% HES (200/0.5) was infused over 1 h starting 30 min after induction of anaesthesia. Horses of the NH group received an equal amount of lactated Ringer's solution (LRS). COP and blood biochemical, cardiopulmonary and anaesthesia-related variables were measured at different time points before and after treatment. Pentastarch was effective in correcting the decrease in COP observed with LRS administration. No differences between treatments were detected for blood glucose, lactate, total proteins and electrolytes. Packed cell volume was lower with the H group immediately after finishing HES-administration and for an additional 30 min. In all horses, all blood biochemical variables other than lactate returned to normal after 12 h. No clinically relevant differences between treatments were detected for cardiopulmonary variables, although 23.1% of the NH-horses needed rescue-HES to maintain cardiovascular function, while none of the H-horses needed additional colloids. Overall, 6 mL/kg HES (200/0.5) was found to be effective in maintaining COP during anaesthesia in systemically healthy horses. Intermediate and long-term effects were below the limit of detection. The potentially beneficial effects on cardiovascular function need further investigation, especially in critically ill horses.