Aging is a complex physiological process characterized by molecular impairments of cell and organ function, increased vulnerability to disease, and eventual demise of the organism. Aging biology has been informed through studies of caloric restriction (CR), defined as a dietary intervention that reduces calorie intake while maintaining proper micronutrient and vitamin nutrition. In the mid-1930s, McCay and coworkers were the first to observe that CR extended the lifespan of rats. More recently, CR has been found to increase the median and maximum of lifespan across a wide range of species including yeast, worm, spiders, flies, fish, mice, rats and perhaps longer-lived species, including monkeys. Although it is unlikely clinical trials will conclusively determine whether CR increases median or maximal lifespan in humans, the Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy (CALERIE) study was designed to investigate the effects of medium-term CR on human aging biology.

The CALERIE study tested the hypothesis that two years of sustained 25% caloric restriction reduces resting metabolic rate and core temperature indicative of metabolic adaptation. Secondary hypotheses included proposed favorable adaptations reflecting primary aging and leading to improvements in aging biomarkers and chronic diseases of aging.

The CALERIE intervention employed an intensive behavioral approach coupled with dietary modifications anticipated to enhance adherence to CR. Intervention staff included psychologists and nutritionists who provided individual and group counseling and utilized a centralized interactive database to monitor interventionists’ practices and participants. Caloric intake was objectively measured using the intake balance method; energy intake was calculated from the difference between changes in energy stores (assessed by dual energy X-ray absorptiometry), and total energy expenditure (TEE) assessed by the doubly-labeled water method. Analyses evaluated the impact of CR on circulating DNA methylation, RNA transcripts, metabolic intermediates and proteins in blood, fat, and skeletal muscle, as well as relating these changes to the intricate biobehavioral measures.

• Age between 21-50 years (inclusive) for men and 21-47 years (inclusive) for women
• Body mass index (BMI) greater than or equal to 22 and less than 28
• Female participants must use acceptable forms of contraception and be willing to continue using such a method while enrolled in the study

• History or clinical manifestation of cardiovascular disease or an elevated blood pressure (greater than 140/90 mmHg)
• Abnormal resting ECG
• History or clinical manifestation of diabetes, cholelithiasis, or any other significant metabolic, hematologic, pulmonary, cardiovascular, gastrointestinal, neurologic, immune, hepatic, renal, urologic disorders, or cancer
• History of anaphylaxis, severe allergies, or asthma
• History of stomach or intestinal surgery (except appendectomy) or major abdominal, thoracic or non-peripheral vascular surgery within one year prior to the randomization date
• Any disease or condition that seriously affects body weight and/or body composition
• Potassium level above the upper limit of normal at the screening visit confirmed by a test repeated within two weeks
• Hemoglobin, hematocrit, RBC, or iron level below the lower limit of normal at the screening visit confirmed by a test repeated within two weeks
• Evidence of active liver disease or ALT levels above 1.5 times the upper limit of normal
• Practice a vegan dietary lifestyle
• History or clinical manifestation of any eating disorder
• Any history of pharmacologic treatment for a psychiatric disorder within one year prior to the randomization date or a history of more than one episode of a pharmacologic treatment for a psychiatric disorder within lifetime
• History of drug or alcohol abuse (up to 14 drinks a week are allowed) within the past two years
• BDI (Beck Depression Inventory) score of 20 or higher at screening or baseline
• Treatment with steroids for more than a month within five years prior to the randomization date, or short-term (less than a month) treatment with steroids within six months prior to the randomization date
• Regular use of other medications, except contraceptives
• Participated in the CALERIE Phase 1 studies or concurrent participation in any other interventional study
• Lost or gained 3 kg or more over the past six months
• Never-smoker of tobacco products or an ex-smoker who quit completely at least 12 months prior to the screening visit
• Donated blood within 30 days prior to the randomization date
• Breast-feeding, pregnant women, or women intending to become pregnant
• Engaged in a regular program of physical fitness involving some kind of heavy physical activity (e.g., jogging, running or riding fast on a bicycle for 30 minutes or more) five or more times per week over the past year
• Unwilling to be assigned at random to the CR or control intervention
• Unable or unwilling to discontinue dietary supplements or adhere to the alcohol consumption restrictions during the study

Participants sustained an average of 12% CR and achieved an average weight loss of ~10% after twelve months of intervention, which was maintained up to the end of the two-year intervention. CR was safe and did not impair the quality of life of the participants and improved mood, sleep, sexual function, and self-reported general health. For primary outcomes, the intervention did not significantly affect core temperature and lowered RMR (adjusted for changes in body energy stores) only marginally and in the first year of the intervention. Weight change and CR caused a lowering of resting metabolic rate more pronounced than expected on the basis of body weight loss (metabolic adaptation), and a decrease in total daily energy expenditure mostly due to decreased physical activity (behavioral adaptation). For secondary outcomes, CR led to significant and persistent inhibition of inflammation without impairing key in vivo indicators of cell-mediated immunity. While CR slightly reduced bone mineral density, this was not in excess of expected changes based on weight. CR improved dietary restraint and self-efficacy, and biomarkers of oxidative stress (e.g., isoprostanes), liver function, and biological aging rate.