Exercise potentially inhibits tumor growth. It remains unclear which processes mediate these effects. Alterations of cytokine concentration in serum can influence cancer cell growth and may cause cell growth inhibition. This pilot study examines whether exercise-induced conditioning in serum can directly affect tumor cells. It focuses on serum collected before and after acute endurance exercise and its impact in vitro. Participants underwent a 1 h endurance training on a cycle ergometer. Samples were collected before, after, and two hours post-exercise. MDA-MB-231 cells were incubated with serum, and cell vitality and proliferation were assessed. Cytokine arrays identified relevant cytokine concentration changes. After identifying CXCL9 as a possible contributor to inhibitory effects, we inhibited the CXCR3 pathway and reassessed vitality. Exercise-conditioned serum significantly reduced cell vitality and proliferation post-intervention and after resting. Cytokine arrays revealed changes in multiple concentrations, and the inhibition of CXCL9 resulted in growth inhibitory effects. Our findings suggest that serum conditioned by an endurance intervention causes changes in cancer cell growth. Based on our observations, the alterations in serum cause growth-inhibitory effects, possibly mediated through the CXCR3 axis. This study provides preliminary evidence supporting the role of exercise in modulating the cancer cell growth directly by changes in serum.