The analysis of cerebrospinal fluid has diagnostic, prognostic, and therapeutic value in neurological illnesses in horses. There are different methods for obtaining cerebrospinal fluid, with the collection between the C1 and C2 vertebrae being a more recent methodology, which allows the procedure to be performed in standing patients, without the limitations of general anesthesia and with a low contamination of the sample with blood, presenting itself as a practical alternative. This study evaluated the efficacy and safety of a local dural blockade in healthy horses submitted to cerebrospinal fluid collection by atlantoaxial puncture and the quality of the samples obtained by this procedure, which were submitted to physical, chemical, and cytological analyses. The animals were evaluated considering aspects such as pain, sensitivity, the presence of edema, temperature variations, and ultrasonographic alterations post-collection. Discrete local changes were observed after the puncture, and the procedure was considered safe and simple to perform. Lidocaine blockade could reduce the reaction elicited by the needle passing through the dura mater, and the samples obtained showed satisfactory quality and laboratory results consistent with the values compiled in the literature. Transient hyperthermia was observed in 70% (7/10) of the animals in the dural blockade group, and 80%(8/10) of the patients from the control group, totalizing 75% of all individuals evaluated. The rectal temperature alteration was observed 4 to 12 hours after the procedure and was entirely resolved without intervention by the 24-hour evaluation.