All subjects completed a comprehensive medical examination, including a detailed self-reported history, physical examination, a resting electrocardiogram, standard blood tests, and an oral glucose tolerance test performed by physicians and nurses in Washington University Clinical Research Unit. Blood tests included: complete metabolic panel, complete blood count, and thyroid stimulating

hormone. Standard cut-offs that are used in the hospital and associated clinics for normal values were used to include or exclude subjects. Selleckchem Androgen Receptor Antagonist For example, the normal ranges for the following blood variables are: white blood cell count, 4.5–13.5 K/μL; red blood cell count, 3.90–5.30 M/μL; hemoglobin, 11.5–16.0 g/dL; thyroid stimulating hormone (TSH), 0.46–4.70 μIU/mL; blood urea nitrogen (BUN), 5–25 MG/dL; blood creatinine,

0.50–1.00 MG/dL; aspartate transaminase (AST), 8–39 U/L; and alanine aminotransferase Tyrosine Kinase Inhibitor Library molecular weight (ALT), 9–52 U/L. Subjects with diabetes, impaired fasting glucose, or impaired glucose tolerance based on American Diabetes Association criteria27 were excluded from the study. None of the subjects had evidence of illness, self-reported insomnia, or were taking medications known to affect sleep or to assist sleep. Their daily caffeine intake was less than 500 mg. VO2peak was evaluated during a graded exercise test on a treadmill. Heart rhythm and rate were continuously monitored (Marquette over MAX-1; ParvoMedics, Sandy, UT, USA) and expired air was analyzed by using a metabolic cart (TrueOne 2400; ParvoMedics). Subjects walked at a constant speed and the inclination of the treadmill was increased by 3% every 2 min until volitional exhaustion and/or two of the following criteria were achieved: respiratory exchange ratio ≥1.15; heart rate greater than the age-predicted maximum (220- age (year)); or plateau in VO2. Each

subject performed two treadmill walking sessions between 9:00 and 11:00 am following an overnight fast. One walking session was at light intensity (45% VO2peak) and one at moderate intensity (60% VO2peak) with randomized sequence, separated by at least 1 week. A snack bar (NatureValley, 250 kcal) was provided before the exercise sessions. The two exercise sessions were performed on same day of the week for each individual to reduce the influence of variation in daily schedule on outcomes. No travel across time zone occurred during the 2 weeks prior to the exercise sessions. During the exercise, expired air was analyzed periodically by using a metabolic cart (TrueOne 2400) to ensure the appropriate exercise intensity was achieved. The duration of exercise was variable and ended when subjects have spent 3.5 kcal (14.7 kJ) energy per kg body weight, based on the volume of the oxygen consumed.