Caloric restriction and intermittent fasting are known to prolong life- and healthspan in model organisms, while their effects on humans are less well studied. In a randomized controlled trial study (ClinicalTrials.gov identifier: NCT02673515), we show that 4 weeks of strict alternate day fasting (ADF) improved markers of general health in healthy, middle-aged humans while causing a 37% calorie reduction on average. No adverse effects occurred even after >6 months. ADF improved cardiovascular markers, reduced fat mass (particularly the trunk fat), improving the fat-to-lean ratio, and increased β-hydroxybutyrate, even on non-fasting days. On fasting days, the pro-aging amino-acid methionine, among others, was periodically depleted, while polyunsaturated fatty acids were elevated. We found reduced levels sICAM-1 (an age-associated inflammatory marker), low-density lipoprotein, and the metabolic regulator triiodothyronine after long-term ADF. These results shed light on the physiological impact of ADF and supports its safety. ADF could eventually become a clinically relevant intervention. Display omitted •For healthy, non-obese adults, ADF is safe to practice for several months•4-week ADF decreases the body weight by 4.5% and improves the fat-to-lean ratio•Cardiovascular parameters and the CVD risk are improved upon ADF•ADF reduces T3 and periodically depletes amino acids, while increasing PUFAs Stekovic et al. show in the clinic that alternate day fasting (ADF) is a simple alternative to calorie restriction and provokes similar improvements on cardiovascular parameters and body composition. ADF was shown to be safe and beneficial in healthy, non-obese humans, not impairing immune function or bone health.