The purpose of the study was to define transient changes in the concentration of inflammatory biomarkers and cartilage biomarkers in the synovial fluid of joints following experimentally induced acute equine synovitis. Acute synovitis was induced in eight skeletally mature mares by a sterile intra-articular injection of 1 mL of phosphate-buffered saline (PBS) containing 0.5 ng of lipopolysaccharide (LPS). The solution was injected into the right middle carpal joint. One mL of sterile PBS was injected into the left control joint. Synovial fluid was obtained at the baseline level and at 8, 24, and 168 h after injection. The levels of inflammatory biomarkers-prostaglandin E2 (PGE2), interleukin 1β (IL-1β), and tumour necrosis factor-α (TNF-α), and cartilage turnover biomarkers-collagenase-cleavage neoepitope of type II collagen (C2C) and C-terminal crosslinked telopeptide type II collagen (CTX-II) were detected with proper assays. Single injections of LPS raised the number of synovial white blood cells and concentrations of total protein, PGE2, IL-1β, TNF-α, C2C, and CTX-II. PGE2 and IL-1β rose sharply at 8 h, while TNF-α increased steadily through 8 h and 24 h, at that point; these three factors returned to the baseline level by 168 h. The time course of C2C and CTX-II concentrations peaked sharply at 24 h, and continued to be significantly elevated over the baseline level even at 168 h. Injections of LPS into the joints led to a temporal inflammatory response, which in turn increased local release of inflammatory biomarkers and significantly altered the concentrations of cartilage markers in the synovial fluid.