Bites of brown spiders (Loxosceles spp.) are responsible for dermonecrotic lesions and potentially systemic envenoming that can lead to death. The only effective therapy is the use of the antivenom, usually produced in horses. However, little is known about the consequences of the systematic use of the Loxosceles venom and adjuvants and of the bleedings on antivenom-producing horses. Thus, the aim of this study was to evaluate the clinical changes in horses in their first immunization protocol for Loxosceles antivenom production. Eleven healthy horses, never immunized, were evaluated in three different periods: T0 (before immunization); T1 (after their first venom immunization); and T2 (after their first bleeding). Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Several suppurated subcutaneous abscesses occurred due to the use of Freund’s adjuvants and thrombophlebitis due to systematic venipunctures for the bleeding procedures. ECG showed arrhythmias in few horses in T2, such as an increase in T and R waves. In summary, the immunization protocol impacted on horses’ health, especially after bleeding for antivenom procurement.