This paper summarizes the findings of the first randomized clinical trial to test whether CR in young, healthy individuals sustained over 2 years provides support for the rate of living and oxidative damage theories of aging.

Out of 80 eligible subjects, 73 consented to the ancillary study and 2 subjects did not complete the baseline metabolic chamber measurement ( Figure 1 ). During the course of the 2-year study, 3 subjects failed to complete all 3 chamber stays (2 in the CR group and 1 in the control) and 7 subjects dropped (6 in the CR group and 1 in the control), leaving a group of 60 subjects with complete data available for analysis. Based on adherence to the prescription (weight change), the final data analyses were performed on 53 subjects (36 females, mean age = 40 ± 6 years), of which 34 subjects were randomized to the CR intervention and 19 subjects to the ad libitum control group. The 53 participants were objectively selected on the basis of adherence to their assigned treatment groups, whereby the criteria for adherence was >5% weight loss at either year 1 (Y1) or year 2 (Y2) for CR participants (3 excluded) and ≤5% weight loss at either Y1 or Y2 for control participants (4 excluded). Per design, all subjects were healthy with 22 (41.5%) being normal weight and 31 (58.5%) slightly overweight at screening. The cohort was 39.8 ± 6.3 years of age at randomization and the majority were female (67.9%) and White (73.6%).

Analyses were performed on 53 men and women who, on the basis of an objective pre-analytical criterion (weight change), were determined to be adherent to their assigned treatment groups.

As anticipated by the study design and use of a mathematical model to guide weight loss during the CR intervention ( STAR Methods ), after Y1, the CR group achieved a 16.5% ± 1.5% reduction in energy intake (or CR) from baseline with an overall 14.8% ± 1.5% CR over the entire 2-year intervention ( Figure 2 A). Despite a slight tendency to gain weight, there was no change in energy intake in the control group from baseline at either time point (Y1, −1.8% ± 2.0% CR; Y2, −1.7% ± 2.0% CR). Subjects in the CR group experienced a significant weight loss at Y1 (−9.4 ± 0.4 kg), which was maintained at Y2 (−8.7 ± 0.4 kg; Figure 2 B). Subjects in the control group essentially maintained weight during the 2-year period ( Figure 2 B). In the CR group, the majority of weight loss (70.7%) was fat mass (Y1, −6.7 ± 0.3 kg; Y2, −5.9 ± 0.3 kg; p < 0.0001 from baseline, within group effect); however, a significant loss in fat-free mass (Y1, −2.9 ± 0.2 kg; Y2, −3.1 ± 0.2 kg; p < 0.0001 from baseline, within group effect) was also observed from baseline at both time points ( Figure 2 B).

Percent of calorie restriction (A) achieved after 1 and 2 years of calorie restriction and the resulting change in fat mass (FM) and fat-free mass (FFM) (B). N = 53; 34 CR, 19 controls. The p value for statistically significant treatment group effects, adjusted for multiple comparisons, is shown. The changes in weight, FM, and FFM were all significantly different between the CR and control group (p < 0.0001 for all, treatment main effect).

Believed to be a mechanism for energy conservation, metabolic adaptation has been the focus of weight loss (not specifically CR) in overweight or obese individuals undergoing intensive dietary interventions (). Weight loss-induced metabolic slowing has been reported in overweight/obese individuals with weight loss maintenance persistent for up to 7 years (). However, the weight loss literature has not focused on quantifying the downstream effects on oxidative damage, which is the other key assumption in a reduced energy flux for delaying biological aging and potentially extending lifespan.

Metabolic adaptation following massive weight loss is related to the degree of energy imbalance and changes in circulating leptin.

In our earlier pilot study, which achieved 19% CR but across only 6 months, we observed a significant metabolic adaptation of EE (24 hr and sleep) measured in a metabolic chamber that was paralleled by a reduced oxidative damage to DNA (). The multi-center CALERIE trial in 218 individuals, using a measure of resting metabolic rate with a bedside indirect calorimeter (ventilated hood), observed a metabolic adaptation after 1 year in comparison with the controls (ad libitum diet), but this adaptation was no longer significant after another year of weight stability ().

Support for the rate of living hypothesis as a mechanism, which may extend lifespan in mammals, is debated (). Indeed, some of this conflict is due to discrepancies in the different degrees and duration of the imposed CR, the timing of the EE evaluation following the initiation of CR, and whether EE was measured under basal or free-living conditions, which includes physical activity. However, probably the most likely source of conflicting evidence is due to conclusions being drawn from EE measurements without appropriate statistical approaches used to account for the losses of metabolic tissues, which also are reduced with CR (). For instance, regarding the observations in rodents, the early seminal work of McCarter and colleagues showed that the basal metabolic rate in rats following a 40% CR for 6 months (), and across the entire lifespan (), was comparable when compared with rats fed ad libitum. These data were, however, soon criticized for the assumption and statistical consideration that oxygen consumption should be adjusted for metabolic mass (). In contrast to McCarter's studies, the evaluation of 24-hr energy metabolism in rats after 11 weeks of moderate (25%) and severe (50%) CR showed a decrease in oxygen uptake (), which, after adjustment for the change in body mass, was only observed in severe CR. In agreement with the necessity for more severe CR in rats, a 14% lowering of the metabolic rate (adjusted for body size) was observed following a 60% CR for 6 weeks, in comparison with counterparts fed ad libitum (). Of the three non-human primate colonies, a 30% CR diet had no impact on oxygen consumption (measured over 36 hr) after 1 year in comparison with controls (). However, EE (night time and 24 hr) examined in the same cohort after 30 months was significantly lower in CR monkeys (). This agrees with a reduction in total daily EE (adjusted for fat-free mass) observed in a different colony undergoing a 26%–31% CR for more 10 years. Yet despite a consistently lower 24hEE in a third colony exposed to a 30% CR, the reduced EE was not different from the control animals ().

An ongoing debate among metabolism, obesity, and aging investigators is whether a chronic deficit in energy intake leads to metabolic slowing or a decreased rate of living. This phenomenon, which has been termed “metabolic adaptation,” defines a reduction in EEs that is larger than expected for a reduction in the respiring mass due to a caloric deficit (). It is thought that the rate of biological aging may be delayed by prolonged CR through a reduction in the rate of living (), leading ultimately to reduced oxidative damage. Together these theories imply that increased metabolism (above what is required to support the respiring mass) and the resultant increased production of ROS lead to a shorter lifespan unless these ROS are removed by antioxidant mechanisms.

Sedentary 24-hr energy expenditure (24hEE) was significantly reduced from baseline in both the CR and control groups at Y1 and Y2 ( Table 1 ), whereas energy expenditure during sleep (SleepEE) was reduced from baseline only in the CR group at both time points ( Table 1 ). In response to the reduced body weight, we observed an approximate 10% drop in absolute SleepEE. After taking the loss of metabolic tissues (fat-free mass and fat mass) into account, SleepEE was still reduced by ∼7% in the CR group, indicating a metabolic adaptation in comparison with the control group ( Figure 3 A; p < 0.02). Similarly, 24hEE adjusted for changes in body composition (fat-free mass and fat mass) was significantly decreased from baseline at Y1 and Y2, but not differently from the control group ( Figure 3 B; p > 0.55).

A comparison of metabolic adaptation in sleep energy expenditure (A) and 24-hr energy expenditure (B) between the AL (control, n = 19, ■) and CR (n = 34, □) groups, after 1 and 2 years of calorie restriction. Metabolic adaptation was considered to represent the change in energy expenditure after adjusting for the changes in fat-free mass, fat mass, age, and sex, and the metabolic adaptation at baseline (see STAR Methods , sedentary 24-hr energy expenditure, for calculation). The p values for statistically significant treatment group effects, adjusted for multiple comparisons, are shown.

Baseline data are presented as means ± SD. Change from baseline data is the adjusted LS mean ± SE from the mixed linear models, which includes the baseline value as a covariate. BMI, body mass index; 24hEE, 24-hr energy expenditure; SleepEE, energy expenditure during sleep (02:00–05:00 hr); T3, triiodothyronine; T4, thyroxine; TSH, thyroid-stimulating hormone.

Physical Characteristics of 53 Men and Women during Weight Maintenance at Baseline and Following 1 and 2 Years of CR

Table 1 Physical Characteristics of 53 Men and Women during Weight Maintenance at Baseline and Following 1 and 2 Years of CR

The lack of effect of CR on physical AREE was echoed in our preliminary 6-month study (). Although the measurement of SPA in the metabolic chamber alludes to a possible reduction in activity with CR, physical activity was unfortunately not objectively measured in the present study while participants were free living.

Recent investigations in non-human primates () allude to increased physical activity at a lower metabolic cost with sustained CR. The energy cost of physical activity, termed activity-related energy expenditure (AREE) was calculated as the cost of daily activities beyond sleep using linear regression model of total daily energy expenditure and SleepEE at baseline ( STAR Methods ). We observed no significant treatment effect for activity-related EE (AREE) (p = 0.20). AREE in the CR group was not changed at Y1, but was significantly decreased from baseline at Y2 (−119 ± 55 kcal/day; p = 0.03, within group effect). In the control group, there was no change in AREE from baseline at either time point. The mean change in spontaneous physical activity (SPA) (kcal/day) measured in the metabolic chamber was not different between the CR and control groups (p = 0.46, treatment main effect). SPA was significantly decreased from baseline in the CR group at both Y1 and Y2 (Y1, −29 ± 9 kcal/day; Y2, −30 ± 9 kcal/day; p < 0.01 for both, within group effect), suggesting a reduced energy cost of activity in the chamber.

Mediators of Energy Metabolism, Biomarkers of Aging, and Relationship with Metabolic Adaptation

Figure 4 Comparison of Changes in Leptin and Thyroxine and the Association between Metabolic Adaptation in SleepEE Show full caption A comparison of changes in the potential mediators of metabolic adaptation, leptin (A) and thyroxine (T4) (B), and the association between metabolic adaptation in SleepEE with percent change from baseline in leptin concentrations at year 1 (Y1) (C) and percent change from baseline in thyroxine concentrations (T4) at year 2 (Y2) (D). AL (control, ■) and CR groups (□). The p values for statistically significant treatment group effects, adjusted for multiple comparisons, are shown. Scatterplots show the linear regression model with 95% confidence interval. N = 53; 34 CR, 19 controls. As expected from the differential changes in fat mass between the treatment groups, there was a significant treatment effect for the change in leptin (p < 0.0001, treatment main effect), with significant reductions from baseline in the CR group at both Y1 and Y2, and no observed changes in the control group ( Figure 4 A). Triiodothyronine (T3) concentrations were significantly decreased from baseline at Y1 and Y2 in the CR group (Y1, −23.5 ± 2.7 ng/dL; Y2, −29.9 ± 2.7 ng/dL; p < 0.0001 for both, within group effect), and that was significantly different from the change in the control group (p < 0.01, treatment main effect). Similarly, there was a significant treatment effect observed for the change in thyroxine (T4) concentrations ( Figure 4 B; p = 0.02), and the post hoc comparison revealed that the change in T4 concentration from baseline in the CR group was only significant at Y2 (p < 0.0001). This reduced activity of the thyroid axis was not supported by a change in the thyroid-stimulating hormone (TSH) (Y1, −0.15 ± 0.09 μIU/mL; Y2, −0.16 ± 0.08 μIU/mL; p < 0.10 for both, within group effect) or reverse T3 (data not shown). Sympathetic nervous system activity assessed through excretion of urinary catecholamines over 24 hr during the chamber was not changed from baseline for epinephrine or norepinephrine in either group at Y1 or Y2. Core temperature recorded over 24 hr was not changed during the weight loss phase (Y1, −0.01°C ± 0.03°C; p = 0.88, within group effect); however, with weight maintenance and sustained CR (Y2), there was a trend for core temperature to be decreased from baseline (Y2, −0.06°C ± 0.03°C; p = 0.07, within group effect). When daytime (08:00–22:00 hr) and nighttime (02:00–05:00 hr) temperatures were considered separately, no change in daytime temperature was observed in the CR or control at either time point and therefore the reduced 24-hr core temperature in the CR group at Y2 was attributed to a reduction in core body temperature recorded at night (Y1, −0.05°C ± 0.05°C, p = 0.30; Y2, −0.10°C ± 0.05°C, p = 0.05, within group effect).

While there was no observed treatment group effect on the changes in fasting concentrations of DHEAS, there was a significant interaction (diet intervention group by time) for fasting insulin (p < 0.05), with a significant reduction in insulin concentration in the CR group at Y1, which was no longer evident at Y2 (Y1, −1.5 ± 0.4 μIU/mL, p < 0.001; Y2, 0.15 ± 0.4 μIU/mL, p = 0.70, within group effect). Furthermore, there was a significant increase in adiponectin concentrations (high molecular weight) from baseline in the CR group (Y1, 1,188 ± 275 ng/mL; Y2, 1,185 ± 271 ng/mL; p < 0.001 for both, within group effect), which was different from control group (Y1, 23 ± 363 ng/mL; Y2, −722 ± 363 ng/mL; within group effect) and at both time points (treatment main effect, p < 0.001).

During the weight loss phase (Y1), the metabolic adaption in SleepEE (SleepEE residual) was associated with greater reductions in leptin ( Figure 4 C; r = 0.35; p = 0.01), but not in the thyroid axis activity (T3, T4, TSH, or reverse T3). At Y2, however, when weight loss was maintained, the relation between metabolic adaptation in SleepEE and leptin was no longer significant (p = 0.22), but the metabolic adaptation was correlated with the reduction in T4 concentrations ( Figure 4 D; r = 0.33; p = 0.02).

In comparison with the control group, significant reductions in hormonal mediators of energy metabolism including leptin and thyroid hormones (T3 and T4), and an increase in adiponectin, were observed in the CR group. However, the relationship of the changes in these hormones to the metabolic adaption differed in relation to CR during weight loss (Y1) compared with CR during weight loss maintenance (Y2). The CR group also demonstrated attenuation in two well-described biomarkers of aging: nighttime core body temperature and fasting insulin.