Abstract Background Intradialytic cycling (IDC) may provide cardiovascular benefits to individuals receiving haemodialysis, but the exact mechanism behind these improvements remains unclear. The primary aim of this study was to investigate the effect of a 6-month programme of IDC on circulating endotoxin (secondary analysis from the CYCLE-HD trial). Secondary aims were to investigate changes in circulating cytokines [interleukin-6 (IL-6), IL-10, tumour necrosis factor-α, C-reactive protein (CRP) and the IL-6:IL-10 ratio] and their associations with physical activity, fitness and cardiovascular outcomes. Methods Participants were randomized to either a 6-month programme of IDC (thrice weekly, moderate intensity cycling at a rating of perceived exertion of 12–14) in addition to usual care (n = 46) or usual care only (control group; n = 46). Outcome measures were obtained at baseline and then again at 6 months. Results There was no significant (P = 0.137) difference in circulating endotoxin between groups at 6 months (IDC group: 0.34 ± 0.08 EU/mL; control group: 0.37 ± 0.07 EU/mL). There were no significant between-group differences in any circulating cytokine following the 6-month programme of IDC. Higher levels of physical activity and fitness were associated with lower levels of endotoxin, IL-6, CRP and IL-6:IL-10 ratio. Conclusions Our data show no change in circulating endotoxin or cytokines following a 6-month programme of IDC. However, higher levels of physical activity outside of haemodialysis were associated with lower levels of inflammation.