Previous studies have shown that the same stressor, depending on intensity, controllability, or duration, can have different effects on the immune system. The purpose of this study was to determine the effect of 10- and 20-min rotation on natural killer (NK) cell activity and also to establish if changes in body temperature, proinflammatory cytokine (IL-1β, IL-6, and TNF-α) levels, and proopiomelanocortin (POMC)-derived peptide (ACTH and β-endorphin) levels parallel the changes in NK cell activity in mice. We found that 10-min rotation significantly increased NK cell activity as compared to both the control (home cage) group and the 20-min-rotation group, while NK cell activity in the 20-min group was not significantly changed compared to the control group. Both 10 and 20 min of rotational stress decreased body temperature and induced significant changes in the proinflammatory cytokine and POMC-derived peptide levels as compared to the control group. The pattern of proinflammatory cytokine expression was quite different between the 10- and 20-min rotation groups. All three proinflammatory cytokines were expressed sequentially (at 0 h after rotation TNF-α, at 6 h IL-1β and IL-6, and at 24 h IL-6) in the 10-min rotation group, while the 20-min rotation group had a small increase in IL-1β (6.7 ± 1.8 pg/ml) at 0 h and increased levels of IL-6 at 6 and 24 h. There was a dissociation of ACTH and β-endorphin expression in both groups resulting in significantly more β-endorphin (p < 0.05) in the 10-min group at 6 h and significantly more ACTH (p < 0.04) in the 20-min group at 6 h. IL-1β and β-endorphin have both been shown to have a direct stimulatory effect on NK cell activity. Therefore, we suspect that the significant increase in both IL-1β and β-endorphin at 6 h in the 10-min-rotation group may be involved in the increased NK cell activity observed at 24 h in the 10-min-rotation group.