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Purpose: Experimental studies have shown that prolonged sitting for 2–8 h can cause changes to vascular and metabolic markers; the response of pro-inflammatory cytokines is relatively unexplored. The purpose of this study is to determine the response of interleukin-8 (IL-8) to prolonged and interrupted sitting. Methods: Healthy participants (n = 24, 21.1 years ± 2.2, 50% female) completed a prolonged sitting session (4 h) and an interrupted sitting session (4 h of sitting with 3 min of walking at 60%HRmax, every 30 min) in random order. Saliva and capillary plasma were collected at the beginning (T1) and at the end of each session (T2). Results: Salivary concentrations of IL-8 increased during the prolonged (T1 median: 22.09 pg/mL, T2 median: 86.18 pg/mL; p = < 0.01, ES − 0.55) and interrupted (T1 median: 22.09 pg/mL, T2 median: 51.99 pg/mL; p = 0.021, ES − 0.34) sessions; however, the increase during interrupted sitting was lower (PS median: 134.4%, range: − 43.96 to 1115.69 and IS median: 50.8%, range: − 75.5 to 356.35; p = 0.011, ES − 0.53). In the sub-sample of males, salivary IL-8 did not increase in the interrupted session (T1 median: 22.09, range: 3.496–699.12, and T2 median: 24.96, range: 5.11–533.5, p = > 0.05, ES − 0.16). No significant findings were observed for IL-8 in the plasma. Conclusion: Prolonged sitting appears to increase concentrations of the pro-inflammatory cytokine IL-8 while interrupting this sitting with short bouts of walking blunts this response. Sex appears to moderate this relationship; however, there appears to be a large amount of individual variability. [ABSTRACT FROM AUTHOR]