Night shift work is a risk factor for viral infection, suggesting that night shift schedules compromise host defense mechanisms. Prior studies have investigated changes in the temporal profiles of circulating cytokines important for priming and restraining the immune response to infectious challenges from night shift work, but not by way of a 24-h constant routine of continuous wakefulness devoid of behavioral or environmental influences. Hence the true endogenous pattern of cytokines, and the combined effect of sleep loss and circadian misalignment on these cytokines remains unknown. Here, 14 healthy young men and women underwent three days of either a simulated night shift or a simulated day shift schedule under dim light in a controlled in-laboratory environment. This was followed by a 24-h constant routine protocol during which venous blood was collected at 3-h intervals. Those who had been in the night shift schedule showed lower mean circulating TNF-α (t13 = -6.03, p < 0.001), without any significant differences in IL-1β, IL-8 and IL-10, compared with those who had been in the day shift (i.e., control) schedule. Furthermore, circulating IL-6 increased with time awake in both shift work conditions (t13 = 6.03, p < 0.001), such that temporal changes in IL-6 were markedly shifted relative to circadian clock time in the night shift condition. These results indicate that night shift work compromises host defense by creating cytokine conditions that initially impede anti-viral immunity (lower TNF-α) and may eventually promote autoimmunity (mistimed rise in IL-6).