BACKGROUND AND AIMS: Routine, periodic fasting is associated with a lower prevalence of coronary artery disease (CAD). Animal studies show that fasting may increase longevity and alter biological parameters related to longevity. We evaluated whether fasting initiates acute changes in biomarker expression in humans that may impact short- and long-term health. METHODS AND RESULTS: Apparently-healthy volunteers (N = 30) without a recent history of fasting were enrolled in a randomized cross-over trial. A one-day water-only fast was the intervention and changes in biomarkers were the study endpoints. Bonferroni correction required p </= 0.00167 for significance (p < 0.05 was a trend that was only suggestively significant). The one-day fasting intervention acutely increased human growth hormone (p = 1.1 x 10(-)(4)), hemoglobin (p = 4.8 x 10(-)(7)), red blood cell count (p = 2.5 x 10(-)(6)), hematocrit (p = 3.0 x 10(-)(6)), total cholesterol (p = 5.8 x 10(-)(5)), and high-density lipoprotein cholesterol (p = 0.0015), and decreased triglycerides (p = 1.3 x 10(-)(4)), bicarbonate (p = 3.9 x 10(-)(4)), and weight (p = 1.0 x 10(-)(7)), compared to a day of usual eating. For those randomized to fast the first day (n = 16), most factors including human growth hormone and cholesterol returned to baseline after the full 48 h, with the exception of weight (p = 2.5 x 10(-)(4)) and (suggestively significant) triglycerides (p = 0.028). CONCLUSION: Fasting induced acute changes in biomarkers of metabolic, cardiovascular, and general health. The long-term consequences of these short-term changes are unknown but repeated episodes of periodic short-term fasting should be evaluated as a preventive treatment with the potential to reduce metabolic disease risk. Clinical trial registration (ClinicalTrials.gov): NCT01059760 (Expression of Longevity Genes in Response to Extended Fasting [The Fasting and Expression of Longevity Genes during Food abstinence {FEELGOOD} Trial]).