Measurement and interpretation of acid–base status are important in clinical practice and among racing jurisdictions to determine if horses have been administered alkalinizing substances for the purpose of enhancing performance. The present study used the physicochemical approach to characterize the daily variation in plasma electrolytes and acid–base state that occurs in horses in the absence of feeding and exercise. Jugular venous blood was sampled every 1–2 h from two groups (n=4 and n=5) of Standardbred horses over a 25 h period where food and exercise were withheld. One group of horses was studied in October and one in December. The time course and magnitude of circadian responses differed between the two groups, suggesting that subtle differences in environment may manifest in acid–base status. Significant daily variation occurred in plasma weak acid concentration ([Atot]) and strong ion difference ([SID]), [Cl−], [K+], [Na+] and [lactate−], which contributed to significant changes in [H+] and TCO2. The night-time period was associated with a mild acidosis, marked by increases in plasma [H+] and decreases in TCO2, compared with the morning hours. The night-time acidosis resulted from an increased plasma [Atot] due to an increased plasma protein concentration ([PP]), and a decreased [SID] due to increases in [Cl−] and decreases in [Na+]. An increased plasma [K+] during the night-time had a mild alkalotic effect. There were no differences in pCO2. It was concluded that many equine plasma electrolyte and acid–base parameters exhibit fluctuations in the absence of feeding and exercise, and it is likely that some of these changes are due to daily variation.