Flow chart of the study design. Body weight and food intake were measured every other day over the seven day pre-trial period. All mice received food ad libitum during the pre-trial period. After the pre-trial period, the mice were divided into three groups (n = 4 mice/group) where the average body weight of each group was similar. The mice in each group were then fed the same diet in different amounts: 1) the standard chow diet unrestricted (UR), 2) the standard chow diet restricted to achieve an approximate 15–20% body weight reduction from the pre-trial weight (R), or 3) the standard chow diet restricted to achieve an approximate 15–20% body weight reduction from the pre-trial weight for a period of three weeks, followed by unrestricted re-feeding for a period of three weeks (R-RF). Each mouse in the R and the R-RF groups served as its own control for body weight reduction as previously described . Based on food intake and body weight during the pre-trial period, food in the R and the R-RF groups was reduced until each mouse achieved the target weight reduction of approximately 15–20%. The study was terminated and plasma was collected for two UR mice and four R mice on day 30, and for the remainder of the mice on day 51.