It is well-established that long-term fasting improves metabolic health, enhances the total antioxidant capacity and increases well-being. MicroRNAs oversee energy homeostasis and metabolic processes and are widely used as circulating biomarkers to identify the metabolic state. This study investigated whether the expression levels of twenty-four metabolism-associated microRNAs are significantly altered following long-term fasting and if these changes correlate with biochemical and redox parameters in the plasma. Thirty-two participants with an average BMI of 28 kg/m2 underwent a 10-day fasting period with a daily intake of 250 kcal under medical supervision. RT-qPCR on plasma small-RNA extracts revealed that the levels of seven microRNAs (miR-19b-3p, miR-22-3p, miR-122-5p, miR-126-3p, miR-142-3p, miR-143-3p, and miR-145-5p) were significantly altered following fasting. Importantly, the expression levels of these microRNAs have been consistently shown to change in the exact opposite direction in pathological states including obesity, diabetes, nonalcoholic steatohepatitis, and cardiovascular disease. Linear regression analyses revealed that among the microRNAs analyzed, anti-inflammatory miR-146-5p expression displayed most correlations with the levels of different biochemical and redox parameters. In silico analysis of fasting-associated microRNAs demonstrated that they target pathways that are highly enriched for intracellular signaling such mTOR, FoxO and autophagy, as well as extracellular matrix (ECM) interactions and cell-senescence. Overall, these data are consistent with a model in which long-term fasting engages homeostatic mechanisms associated with specific microRNAs to improve metabolic signaling regardless of health status.