In this study, gastroscopy was performed twice in 31 two-year-old Thoroughbred racehorses in training. Salivary cortisol (SC) and fecal cortisol metabolite (FCM) concentrations were determined to measure the stress impact. One gastroscopy was performed at the beginning and one six months later at the end of the horses’ first training season. According to the Wilcoxon signed rank test, a mild but significant increase of SC (1st gastroscopy: p = 0.0045, 2nd gastroscopy: p < 0.0001) and FCM (1st gastroscopy: p < 0.0001, 2nd gastroscopy: p = 0.0006) values after gastroscopy compared to basal values was detected.