Dark bars represent the scheduled sleep episodes. Subjects completed a 39-day protocol with a baseline ‘sleep replete’ condition with 3 weeks of 10 h/day of time in bed (TIB) at home, then 6 days with ≥10 h TIB per day. Sleep opportunities were then spread across the circadian cycle on a 28-h “forced desynchrony” (FD) protocol, with 6.5 h TIB (equivalent to 5.6 h per 24h) and 21.5 h of monitored wakefulness for 3 weeks. A subsequent period of 10 days of circadian re-entrainment with sleep recovery (10 h TIB/24 h) with the sleep period adjusted to the same circadian phase as the baseline sleep condition by modification of the duration of the wake period after the last FD day. Standardized breakfast meal responses (B); daily fasted blood samples for assessment of glucose, insulin, cortisol (F); core body temperature minimum (white X). Time from midpoint of sleep to start of breakfast (grey horizontal arrow) was maintained by choosing a day in the last week of sleep restriction plus circadian disruption such that the standardized meal occurred during this exposure at the same circadian phase as baseline within 4 h (0.7 ± 1.8 h), resulting in an average exposure duration of 19.2 ± 2.8 24-h days (range 15-22 days).

In young (panels A-D) and older subjects (panels E-H), mean profiles (± 95% C.I) are depicted for glucose (panels A, B, E, F) and insulin (panels C, D, G, H) responses to an identical, standardized breakfast (striped horizontal bar at time=0) under conditions of baseline sleep replete (≥10 h TIB/24 h [dashed black line]), history of prolonged sleep restriction combined with circadian disruption (5.6 h TIB/24 h [solid red line; left panels]), and following 9 days of stable circadian re-entrainment and recovery sleep (10 h TIB/24 h [solid grey line; right panels]). In each condition, breakfast was served at the same circadian temperature phase ± 4 h (0.7 h ± 1.8 h).

Young and older participants were assessed during the conditions of baseline sleep replete (10 hTIB/24 h [B, black bars]), following an average of 19 days of sleep restriction combined with circadian disruption (5.6 h TIB/24 h [SRCD, red bars]), and after 9 days of stable circadian re-entrainment and recovery sleep (10 h TIB/24 h [R, grey bars]). In each condition, a fasted sample was collected prior to an identical breakfast and assayed for glucose (panel A), insulin (panel B), and leptin (panel G). For an hour after the identical breakfast, samples were taken every 10 minutes, and another at 90 minutes post meal and assayed for glucose and insulin. Peak (panels C-D) and area under the curve (AUC) values (panels E-F), were calculated over the first 90 postprandial minutes. Resting metabolic rate (panel H) was determined prior to the meal. Insulin and leptin were log-transformed prior to statistical testing. Values are means ± SE. Bonferroni-adjusted P-values were based on mixed-effects models with age, condition, and age*condition (and gender for RMR) as the fixed effects and participants as the random effects, and are depicted as follows; p≤0.05 *; p≤0.01 **; p≤0.001 ***; p≤0.0001 ****. Bonferroni adjustments were applied to each age group separately.

Mean fasted levels (±95% CI) of glucose (A), log insulin (B), cortisol (C) from samples collected within an hour of awakening at all circadian phases and post-void body weight (D). For reference, the mean level (±95% CI) of the fasted value at baseline for each measure is depicted at the approximate circadian phase of the baseline assessment (black circle). Circadian phase of the sample collection was determined using core body temperature recordings (see Methods). There was a significant circadian variation, but no significant effects of age for all measures. Week 1 differed from week 3 for insulin levels and weight. Significant p values are depicted as follows; p≤0.05 *; p≤0.01 **; p≤0.001 ***; p≤0.0001 ****.

Free ghrelin and leptin response to a meal in young and older participants at baseline, following an average of 19 days of prolonged sleep restriction combined with circadian disruption, and following 9 days of stable re-entrainment and recovery sleep. In young (panels A, C, E, G) and older subjects (panels B, D, F, H), mean profiles (± 95% C.I) are depicted for free ghrelin (panels A-D) and leptin per percent body fat (panels E-H) and aligned relative to an identical, standardized breakfast (time=0) under conditions of baseline sleep replete (≥10 h TIB/24 h [dashed black line]), history of prolonged sleep restriction combined with circadian disruption (5.6 h TIB/24 h [solid red line]), and following 9 days of stable circadian re-entrainment and recovery sleep (10 h TIB/24 h [solid grey line]). In each condition, breakfast was served at the same circadian temperature phase ± 4 h (0.7 h ± 1.8 h). Sleep periods are depicted by horizontal bars, meals by vertical bars.