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Electroejaculation (EE) is a technique widely used to collect semen in ruminants, which produces a stress response with negative effects on animal welfare. The aim of this paper was to characterize the stress response during conventional EE in rams that have been frequently electroejaculated. Blood samples were collected since 20 min before and until 120 min after electroejaculating 10 rams. Electroejaculation affected hormone concentration, as cortisol increased, and testosterone decreased after EE. Heart and respiratory rate increased 10 min after EE. Several blood parameters, such as glycaemia, an increase in total protein and creatine kinase concentrations and a decrease in haematocrit, haemoglobin, red blood cell and alkaline phosphatase concentrations. The rams vocalized 13.8 ± 2.4 times, and the largest length of vocalizations during EE was associated with the numbers of pulses in which the rams ejaculated. In this paper, we provide a complete characterization of the stress response to EE in rams. We showed that although rams have been frequently electroejaculated, an important stress response including changes in respiratory rate, testosterone concentrations, haematological and biochemical parameters were observed, besides the changes in cortisol concentrations and heart rate, suggesting that rams were not habituated to EE. In addition, we described the main parameters of vocalizations and its relation with the moment of EE. The information displayed on the frequent use of EE in the rams should be considered in relation to the welfare implications.