This study examined how a standard exercise test (SET) affected (1) thyroid hormones (THs) of horses and (2) the relationship between the VLa4 of horses and TH responses to the exercise in trained Standardbred racehorses (VLa4 is the velocity run at defined conditions at which a blood lactate concentration of 4 mmol/L is determined). Twelve trained Standardbred racehorses (six stallions and six mares) performed SETs until the horses' blood lactate concentration was at or above 4 mmol/L. The horses were divided into three age groups (2, 3, and 4 years old); each group consists of 4 horses respectively (two male and two female), to evaluate the effects of age and sex on hormonal responses to SET. During each SET, blood samples were taken at rest and after each interval and at the end of SET. Blood was analysed for total and free triiodothyronine (T3, fT3) as well as total and free thyroxine (T4, fT4). The statistical model included three fixed factors (SET, sex and age) and their main interactions. ANOVA analysis revealed that T3 and fT3 were significantly influenced by SETs. Plasma T3 and fT3 concentrations were higher in 4-year-old horses compared to the other age groups. All plasma THs concentrations were higher in mares than in stallions. Correlations revealed that a higher VLa4 was negatively related to all THs responses in 2-year-old Standardbred mares only. The SET used to determine VLa4 increased selected THs (T3, fT3); these increases were inversely related to VLa4 and affected by age and sex of the horses. The correlation of VLa4 with thyroid exercise' response might provide some additional information for performance evaluation of Standardbred racehorses, especially for evaluating training adaptation, according to sex, and age. Further studies are necessary to provide support on the value of measuring THs in Standardbred racehorses of different sex and age.