In the last decades, a well-established equine wound model has been used to study fibroproliferative wound healing disorders. The aim of this study was to characterize the degree of discomfort of wounding and sampling in an equine excisional wound model by evaluating systemic and local inflammatory responses and signs of pain. A total of 12 cutaneous wounds, three on each shoulder and each metatarsus, were created in a standing surgical procedure. Wounds were biopsied on days 2, 4, 7, 14, 21, and 28 after surgery. Clinical parameters (rectal temperature, heart rate, respiratory frequency) and blood levels of white blood cell, serum amyloid A, fibrinogen, and iron were monitored to evaluate the systemic inflammatory response. Local signs of inflammation (swelling, heat, pain) were subjectively assessed, the limb circumference recorded, and temperature of the wound measured by thermometry. Pain was evaluated by a composite measure pain scale (CMPS). The results demonstrated that the wounding procedure elicits an inflammatory response. Day 1 after surgery, two horses scored 2 and 7 units (of 27 units), respectively, on the CMPS, and day 8 after surgery, one horse scored 3 units. The biopsy procedure did not elicit local or systemic signs of inflammation. Based on these findings, it appears that the equine experimental wound model causes mild discomfort and pain manifestations. This information is important for researchers, who consider using the model. To justify the use of an animal model, it should be demonstrated that the expected benefits of the research outweigh the discomfort imposed to the animal.