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The objective of this study was to investigate the impact of heat stress on the expression of estrus signs and follicular diameter at the day of estrus in dairy cows under farm conditions. Cows reported in estrus (i.e., by an automated activity monitoring system or by the herd manager) were examined by a veterinarian. Uterine contractility and the largest diameter of all ovarian structures was determined by transrectal palpation and ultrasonography, respectively. The amount of estrus discharge, mounting traces and the color of the vaginal mucosa were determined in an external examination and scored on 3-point scales. Blood samples were obtained for analysis of serum progesterone concentrations. Cows with a high uterine contractility and high amount of estrus discharge were 4.05 and 1.72 times more likely to have an estrus follicle (large, presumptive preovulatory follicle ≥ 12 mm) than cows expressing low amount of the estrus sign, respectively. The likelihood for a pink vaginal mucosa, clear stringy estrus discharge and mounting traces at the cows back decreased continuously with increasing temperature-humidity index (THI) at the day of estrus. The likelihood for a serum progesterone concentration < 1 ng/ml at the day of estrus decreased continuously with increasing THI ≥74. Follicular size decreased 0.1 mm for each incremental THI point at the day of estrus. The results of this study indicate, that heat stress at the day of estrus significantly reduces the intensity of external estrus signs and the size of estrus follicle decreases with increasing THI.