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Summary: Background: There are currently no studies detailing cardiac troponin I (cTnI) release in normal horses post‐exercise using an analytically validated assay. These data are essential for selecting appropriate sampling times in equine athletes with suspected myocardial injury. Objective: To plot the magnitude and time course of cTnI release after maximal effort, using validated cTnI assays. Study design: Descriptive longitudinal study. Methods: Five clinically normal Standardbred racehorses in race training were included in the study. Horses were exercised in harness at near‐race intensity. Blood samples were taken immediately pre‐ and post‐exercise and then hourly for 24 h. Samples were analysed using the validated high‐sensitivity cTnI assay and a contemporary sensitivity cTnI assay. Results: Mean resting cTnI was 1.33 ± 0.6 s.d. ng/L (range, 0.82–2.33 ng/L) using assay A. All horses were below the detection limit at rest using assay B. Peak elevation occurred 2–6 h post‐exercise with both assays (mean, 4.6 ± 1.7 and 4.0 ± 2 h, respectively). Mean peak increase in cTnI was 11.96 ± 9.41 ng/L (range, 1.72–23.76 ng/L) using assay A. Peak concentrations were detectable in three of the horses using assay B and were between 0.039 and 0.051 μg/L (mean: 0.043 ± 0.006 μg/L). All horses returned to baseline within 24 h. Main limitations: A small (n = 5) convenience sample was used as random sampling was not logistically possible. Conclusions: All horses experienced an increase in cTnI post‐exercise, with peak occurring 2–6 h post‐exercise. Cardiac troponin I elevation was detected earlier using the high‐sensitivity assay, which may convey a diagnostic advantage. Targeted studies are needed to determine the significance of these increases. [ABSTRACT FROM AUTHOR]