In terms of exercise, testosterone is important for the growth and maintenance of skeletal muscle mass. Sampling saliva could be a non-invasive alternative to blood sampling for the quantification of testosterone levels in horses. The objective of this study was to compare testosterone concentrations in saliva and serum (sampled simultaneously) from horses of different sexes and at different times throughout the day. A total of 67 warmblood riding horses (21 geldings, 22 mares and 24 stallions) were included in the study. Saliva and blood samples were collected in the morning (06:00-08:00), at midday (11:00-13:00) and in the evening (17:00-19:00). The results demonstrated a weak correlation between saliva and serum testosterone concentrations (r_s=0.25, P=0.04). Stallions had higher serum testosterone concentrations than mares and geldings (P<0.001), but there was no significant effect of sex on salivary testosterone concentrations. The time of day did not affect the concentration of testosterone in either saliva or serum. In conclusion, our results indicate that saliva samples cannot be recommended for measuring testosterone levels in horses. However, further research is needed to identify the disturbing factors.