Objectives. The acute fasting-induced cardiovascular autonomic response and its effect on cognition and mood remain debatable. Thus, the main purpose of this study was to estimate the effect of a 48 h, zero-calorie diet on autonomic function, brain activity, cognition, and mood in amateur weight lifters. Methods. Nine participants completed a 48 h, zero-calorie diet program. Cardiovascular autonomic function, resting frontal brain activity, cognitive performance, and mood were evaluated before and after fasting. Results. Fasting decreased (p < 0.05) weight, heart rate, and systolic blood pressure, whereas no changes were evident regarding any of the measured heart rate variability indices. Fasting decreased (p < 0.05) the concentration of oxygenated hemoglobin and improved (p < 0.05) mental flexibility and shifting set, whereas no changes were observed in working memory, visuospatial discrimination, and spatial orientation ability. Fasting also increased (p < 0.05) anger, whereas other mood states were not affected by it. Conclusions. 48 h fasting resulted in higher parasympathetic activity and decreased resting frontal brain activity, increased anger, and improved prefrontal-cortex-related cognitive functions, such as mental flexibility and set shifting, in amateur weight lifters. In contrast, hippocampus-related cognitive functions were not affected by it.