Magnesium (Mg) and calcium (Ca) are essential electrolytes that are involved in various physiological actions in the body. The alterations of these electrolytes’ homeostasis can lead to their deficiency, which can adversely affect their various essential functions. Sleep deprivation is known to be a form of stress, and it can alter behavioral, physiological, as well as cellular functioning; therefore, stressor-like sleep deprivation may affect the levels of Mg and Ca. We examined the plasma electrolytes in a total of 18 male Sprague–Dawley rats that were divided equally into three groups. Blood samples were collected after each experimental protocol and were analyzed for plasma levels of Mg2+ and Ca2+, and the total plasma Mg (bound + free Mg2+) and Ca (bound + free Ca2+), respectively. Sleep deprivation significantly (P < 0.05) decreased the plasma levels of free Mg2+ and Ca2+. In contrast, there was no significant difference in ionized plasma Ca and Mg concentrations in sleep-recovery and control rats. There was also no significant difference in the total plasma Ca and Mg concentrations between the groups. Analyses of sleep stages indicated a sleep-rebound effect, with a significant increase in both rapid eye movement (REM) and non-REM sleep stages in sleep-recovery rats. The results show that sleep deprivation might be one of the contributing factors causing the loss of Mg and Ca electrolytes and the problems associated with the integrity of physiological system functions caused by sleep loss.