Objective-To explore diurnal variation in blood ionized calcium, sodium, potassium, and chloride concentrations and pH in pregnant dairy cows. Animals-14 dairy cows in their third or later pregnancy approximately 6 weeks before expected parturition. Procedure-Throughout a 24-hour period, blood samples were taken at 2-hour intervals and analyzed for ionized calcium (iCa(2+)), potassium (K+), sodium (Na+), and chloride (Cl-) concentrations and pH. Paired t-tests were used to compare initial and final values. Circadian changes were tested by use of repeated-measures ANOVA. Additionally, a nonparametric analysis was performed for each animal to determine minimal and maximal values for the variables. Results-Significant differences were not detected between initial and final values of any variable. Repeated-measures ANOVA indicated significant diurnal fluctuations in all variables. With the exception of Cl- concentration, nonparametric analysis of individual values also revealed significant changes over time; Conclusions and Clinical Relevance-The magnitude of daily fluctuations does not make it necessary to standardize time of sample collection for routine diagnostic examination of blood iCa(2+), Na+, K+, and Cl- concentrations and pH. However, it may be important to standardize time of sample collection in comparative studies in which more discrete differences may be identified.