Background: Autologous conditioned serum (ACS) is a commonly administered intra-articular treatment for the management of osteoarthritis in athletic horses. Objectives: To investigate the influence of exercise on the concentration of cytokines in a non-commercial method of ACS production. Study design: Non-randomised cross over design. Methods: Whole blood was obtained from eight healthy Standardbred horses immediately prior to, 1 h and 24 h following a single bout of exhaustive exercise. Blood was processed using a non-commercial method of ACS production. Fluorescent microsphere immunoassay (FMIA) analysis was performed to quantify Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin 1β (IL-1β) and tumour necrosis factor α (TNF-α) concentrations at each time point. Mixed effect repeated measures analysis of variance (ANOVA) was used to compare the pre-exercise and post-exercise cytokine concentrations. Significance was set at P < 0.05. Results: A reduced concentration of IL-1Ra (median 584.4, IQR 81.9–5098 pg/ml, p = 0.004) and an increased concentration of TNF-α (11.92, 9.28–39.75 pg/ml, P =.05) at 1 h post-exercise were observed when compared with baseline values (IL-Ra 7349, 1272–10 760 pg/ml; TNF TNF-α 11.16, 8.36–32.74 pg/ml). No difference in cytokine concentrations of IL-10 or IL-1β were found between any of the time points. Main limitations: The large biological variability and small sample size represents limitations of this study. Conclusions: These results suggest that a single bout of intense exercise can reduce the concentration of the anti-inflammatory cytokine IL-1Ra and increase the concentration of the pro-inflammatory cytokine TNF-α, reducing the 'anti-inflammatory' cytokine composition of ACS. Our findings suggest that collection of blood for ACS production should be performed no sooner than 24 h following a single episode of intense exercise.