BACKGROUND: One acute bout of exercise leads to a rapid increase in the systemic cytokine concentration. Regular exercise might alter the cytokine response, in particular in beforehand untrained and obese subjects. METHODS: Using a proximity extension assay, we studied the effects of acute exercise as well as endurance training on a panel of 92 cytokines related to inflammation. 22 subjects (30 ± 9 years; VO2peak 25.2 ± 4.2 ml/(kg*min); BMI 31.7 ± 4.4 kg/m2) participated in an 8-week endurance exercise intervention. Blood samples were collected before and immediately after 30 min ergometer exercise at 80% VO2peak. RESULTS: Before and after the training intervention, 40 and 37 cytokines, respectively, were acutely increased more than 1.2-fold (BH-adjusted p < 0.05). The exercise intervention did not change the acute increase in cytokines nor the resting cytokine levels, whereas fitness was improved and adiposity reduced. The increase in fitness led to a slight increase in power output when exercising at the same heart rate, which might explain the comparable increase in cytokines before and after the intervention. The largest acute increase was found for OSM, TGFA, CXCL1 and 5 and TNFSF14 (≥ 1.9-fold, BH-adjusted p-values < 0.001). The transcript levels of these proteins in whole blood were also elevated, particularly in the trained state. Only the acute increase in IL6 (1.3-fold) was related to the increase in lactate, confirming the lactate-driven secretion of IL6. CONCLUSION: Our comprehensive proteomics approach detected several underexplored serum exerkines with up to now less understood function in the adaptation to exercise.