Interleukin-6 (IL-6) is a cytokine that can mediate numerous biological actions including fatigue. Circulating IL-6 increases during prolonged exercise, and furthermore, the signalling receptors sIL-6R and sgp130 are also increased. The variability of the response of these markers to exercise is unknown; therefore, we examined the changes in these markers to a preloaded time trial bout of running. Nine males performed three identical trials where participants ran at 60% v[(V)\dot]\textO 2 \textmax vVO2maxfor 2 h interspersed with 30 s at 90% v[(V)\dot]\textO 2 \textmax vVO2maxevery 10 min, followed by a 5-km time trial. Blood samples were drawn at baseline, following the 2-h bout, post time trial, 1 h post time trial and the following morning. Results showed that between-subject variability (CVg) was greater than within-subject variation (CVi) for the three markers. IL-6, sIL-6R and sgp130 demonstrated a CVi of 15.3–25.5%, 15.0–17.6% and 6.2–9.4% variation, respectively, across the time points. When the data from the second and third trials were analysed independently, CVi was reduced which is supported by the time trial results for which CVi improve (4.7–2.4%). In conclusion, the results indicate that a large variation in response to exercise can be reduced following a habituation trial.