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We hypothesised that shorter treatment intervals of intraarticular autologous conditioned serum (ACS) injections would more beneficially affect the synovial fluid (SF) concentrations of IL-1ra, IL-1β and cartilage biomarkers, compared with the traditional weekly treatment intervals in joints suffering from natural OA. In a randomised comparative study, 12 horses with OA were allocated to two groups (n = 6). The horses in group 1 received three intraarticular ACS injections at weekly intervals, whereas the horses in group 2 received three intraarticular ACS injections at two-day intervals. The levels of IL-1ra, IL-1β, CPII, C12C and CS 846 were determined in SF before and after ACS treatment using commercially available ELISA kits. The SF IL-1ra concentration 1 hour and 4 hours after ACS injection was significantly increased compared to baseline levels and decreased back to it within 48 hours. Comparing the SF IL-1ra, IL-1β, C12C, CS 846 and CP II levels before and 42 days after ACS treatment, group 2 showed a significant decrease in all parameters and an approximation on the levels in normal joints. These results indicate that the long-time effect of an ACS treatment given at two-day intervals is characterized by decreased SF IL1ra, IL-1β, C12C, CP II and CS 846 concentrations, which might indicate an improvement in joint inflammation and cartilage degrading processes . Further investigations with greater sample sizes have to prove if the two-day treatment interval is preferable to the widely used treatment protocol of weekly intraarticular ACS injections.