Food intake is regulated by not only neurohormonal, but also social, educational, and even cultural factors. Within the former, there is a complex interaction between orexigenic (ghrelin) and anorexigenic (glucagon-like peptide-1 (GLP-1)) factors in order to adjust the food intake to caloric expenditure; however, the number of subjects that are unable to properly balance appetite and body weight is increasing continuously. A loss of circadian or diurnal coordination of any of these factors may be implied in this situation. Special attention has retrieved GLP-1 due to its usefulness as a therapeutic agent against obesity and related alterations. Thus, the objective of the present study was to compare GLP-1 diurnal synthesis between normal weight and overweight/obese subjects, and to evaluate whether weight loss can restore the synthesis rhythms of GLP-1. Three groups of 25 subjects were divided attending to their body mass index (BMI) in normal weight, overweight, or obese subjects. Diurnal (5 points) GLP-1 levels were analyzed. Secondly, an intervention (behavioral-dietary treatment) study was conducted to analyze the effect of weight loss on plasma GLP-1 concentrations. Our results showed that baseline GLP-1 level was significantly lower in normal weight subjects (p = 0.003); furthermore, our cosinor analysis revealed a higher amplitude (p = 0.040) and daily GLP-1 variation (47%) in these subjects. In fact, our ANOVA data showed a lack of rhythmicity in overweight/obese patients. Weight loss was not able to restore a diurnal rhythm of plasma GLP-1 levels. In summary, the present work shows a disruption of diurnal GLP-1 levels in overweight/obese subjects, which worsen as body fat progresses. The attenuation of the GLP-1 synthesis rhythms may be important to understand the impairment of food intake regulation in overweight/obese subjects.