Although immune dysfunction by space conditions has been reported postflight, as well as during ground-based experiments, the cause(s) and nature of the immunological changes are not completely understood. Microgravity has been suggested as one of the factors responsible for the observed immune dysregulation. The goal of this study was to assess immune changes in simulated microgravity (s-mG) using an in vitro cytokine release assay. The effect of s-mG provided by the desktop random positioning machine on cell-mediated immunity was examined by analyzing interleukin 2 (IL-2), interferon-g (IFN-g), tumor necrosis factor alpha (TNF-a), and interleukin 10 (IL-10), in response to immune cell stimulation in whole blood samples (n = 10). Stimuli used were bacterial recall antigens, pokeweed mitogen (PWM), lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM). S-mG caused an overall inhibition of the IL-2 and IFN-g responses to recall antigen and mitogen stimulation. More specifically, s-mG most strongly influenced the levels of all four cytokines elicited by bacterial recall antigen stimulation. In contrast, HKLM-induced TNF-a secretion was elevated. The average concentrations of TNF-a in response to PWM and LPS and IL-10 release stimulated by PWM, LPS, and HKLM were not significantly altered by s-mG. However, a variable response between individual subjects could be observed. In conclusion, our results demonstrate that the in vitro cytokine release assay can detect gravity-related immune alterations. Furthermore, the use of multiple stimuli and the associated changes in cytokine secretion has the potential to reveal information on the underlying mechanisms affected by s-mG.