The goal of the present study was to investigate the daily pattern of serum cortisol rhythm in relation to the total locomotor activity in horses housed in an individual box under a 12-hour light-dark cycle. Blood samples were collected at 3-hour intervals over a 48-hour period via an intravenous cannula inserted into the jugular vein in 5 Italian Saddle horses. In addition, the horses were equipped with Actiwatch Mini, actigraphy-based data loggers that record a digitally integrated measure of motor activity. The application of cosinor rhythmometry showed a daily rhythmicity of serum cortisol levels and locomotor activity. Both parameters showed a diurnal acrophase, at the beginning of photophase for cortisol anticipates of about 5 hours with respect to the acrophase of locomotor activity. The data on the distribution of acrophases have implications for the issue of causation. In conclusion, we found no influence of the rhythm of locomotor activity with the rhythm of serum cortisol.