ObjectivePhase 1: to determine the feasibility of desensitizing ventral branches of spinal nerves within the rectus sheath using an ultrasound-guided rectus sheath block (USRSB). Phase 2: to determine the effect of preoperative USRSB on intraoperative responses to surgical stimulation and postoperative pain. Study designCadaveric study and prospective, randomized, blinded, parallel-arm clinical trial. AnimalsA group of five cat cadavers and 37 shelter-owned cats undergoing ovariohysterectomy. MethodsPhase 1: anatomical dissection was performed on one uninjected cadaver. Abdominal walls were dissected in four cadavers (eight hemiabdomens) following bilateral USRSB using 1:1 new methylene blue and 0.5% bupivacaine (0.8 mL kg–1 total). Phase 2: preoperative bilateral USRSB was performed with 0.8 mL kg–1 of 0.25% bupivacaine (RSB) or equivalent volume of 0.9% saline (CONTROL). Intraoperative systolic arterial blood pressure (SAP), heart rate (HR), respiratory rate (fR) and vaporizer setting (vap%) were recorded before skin incision, during celiotomy and abdominal wall closure. In recovery, cats were administered robenacoxib (2 mg kg–1; CONTROL) or 0.9% saline (0.1 mL kg–1; RSB) subcutaneously. Postoperative pain was evaluated for 6 hours using the Glasgow Composite Measure Pain Scale. ResultsPhase 1: spinal nerves T9–L3 were identified within the rectus sheath, and stained in 0%, 40%, 63%, 75%, 100%, 88%, 50% and 13% of hemiabdomens, respectively. Phase 2: 37 cats were included (RSB, n = 17; CONTROL, n = 20). Intraoperatively, SAP, HR and fR were not significantly different between groups. Vap% was significantly lower in RSB during celiotomy (p = 0.036) and closure (p = 0.044). Postoperatively, RSB cats were 5.3 times (95% CI 1.8–8.3) more likely to require rescue analgesia than CONTROL cats. Conclusions and clinical relevanceDuring surgery, USRSB with bupivacaine offered minor benefits and provided markedly less postoperative analgesia than robenacoxib, indicating that relying on USRSB provides insufficient postoperative analgesia for ovariohysterectomy in cats.