In this study the authors evaluated the influence of physical activity on the daily rhythm of rectal temperature and total locomotor activity in untrained and trained horses. Rectal temperature and locomotor activity of 12 Italian saddle horses, 6 untrained (group A) and 6 trained (group B), was recorded for 48 h. Rectal temperature was recorded every 4 h with a rectal probe. Animals were equipped with actigraphy-based data loggers, Actiwatch-Mini® to record total activity. The application of two-way ANOVA showed a highly significant effect of time and exercise on rectal temperature in untrained and trained horses in both days of monitoring. A significant effect of time on locomotor activity was observed, but there was no effect of exercise. Cosinor analysis identified the periodic parameters and their acrophases during the 2 days of monitoring. For all rhythmic parameters of rectal temperature except amplitude, no statistically significant differences were observed between the two groups. Statistically significant differences for the rhythmic parameters of locomotor activity, except robustness, between untrained and trained horses were found. In conclusion, rectal temperature circadian pattern was similar in untrained and trained horses, indicating that the endogenous nature of its rhythm was not influenced by external stimuli such as physical exercise.