The physiological, cognitive state, and motor behavior changes that occur during acute fasting are not completely understood. Thus, the aim of this study was to estimate the effect of 2-day total fasting on evoked stress, mood, brain activity, and cognitive, psychomotor, and motor function in overweight women. Eleven overweight women (body mass index above 25 kg/m2) aged 20–30 years were tested under two conditions allocated randomly: 2-day zero-calorie diet with water provided ad libitum and 2-day usual diet. One week before the experiment, aerobic fitness was evaluated. Subjective stress ratings in relation to the diet, autonomic function, prefrontal cortex activity, cognitive performance, psychomotor coordination, and grip strength were evaluated before and after each diet. The study demonstrated that fasting decreased log-transformed high-frequency (HF) power, without affecting heart rate. The relative maximum oxygen uptake was negatively correlated with subjective stress rating and changes in log-transformed HF. Fasting did not affect mood, brain activity, and cognitive, motor, and psychomotor performance. Thus, 2-day total fasting evoked moderate stress with a shift of the autonomic nervous system balance toward sympathetic activity in overweight women. Better aerobic endurance is likely to facilitate the capacity for dealing with acute fasting. Regardless of the evoked stress, cognitive state and motor behavior remained intact.