Circadian Misalignment Augments Markers of Insulin Resistance and Inflammation, Independently of Sleep Loss

Rachel Leproult; Ulf Holmbäck; Eve Van Cauter


Diabetes. 2014;63(6):1860-1869. 

In This Article

Research Design and Methods

Protocol and Participants

The protocol (Figure 1) was approved by the University of Chicago Institutional Review Board, where all participants were studied after giving written informed consent.

Figure 1.

Schematic representation of the study design. The protocol followed a parallel group design with two experimental interventions: sleep restriction with circadian alignment (left) and sleep restriction with circadian misalignment (right). The black bars represent the periods allocated to sleep. In both groups, 3 baseline days of 10-h bedtimes (from 2200 to 0800 h; B1, B2, B3) were followed by 8 days of sleep restriction to 5-h bedtimes (R4–R11). In the circadian alignment group, all short sleep periods were centered at 0300 h (bedtimes: 0030 to 0530 h). In the circadian misalignment group, four of the eight short sleep periods (R5, R6, R8, and R9) were delayed by 8.5 h, such that sleep occurred during the daytime (0900 to 1400 h). In both groups, breakfast (B) was served between 0730 and 0830 h, lunch (L) between 1300 and 1400 h, and dinner (D) between 1900 and 1930 h. On shifted days in the misalignment group, lunch was served at 1500 h, 1 h after wakeup time, and a sandwich (S) was presented at 0100 h. Snacks were available at all times. An IVGTT was performed at 0900 h on B2 and on R10. Two 24-h sessions of blood sampling at 15- to 30-min intervals were performed on B3 and R11 (dashed lines). Caloric intake during these sessions was limited to three identical carbohydrate-rich meals (HC). No snacks were allowed. Saliva sampling at 30-min intervals was performed from 1600 to 0030 h on R4 and R11 to assess melatonin levels (gray bars).

We compared two 11-day interventions using a parallel group design. For logistic reasons primarily related to staffing, the two interventions could not be conducted simultaneously. Therefore, the participants were not formally randomized but assigned to the intervention that was implemented at the time of their recruitment, without being aware that there was an alternate intervention.

Participants from the local community responded to advertisements inviting healthy adults with normal body weight and ages 21–39 years to participate in a research study, "Extended work schedules and health: Role of sleep loss" and involving 2 weeks of hospitalization. Supplementary Figure 1 shows the flow diagram of subject recruitment and participation. Participants underwent a physical examination and laboratory tests to rule out endocrine, psychiatric, and sleep disorders; medication use; smoking; excessive alcohol or caffeine consumption; shift work or travel across time zones during the past 2 months; and self-reported habitual sleep of less than 7.5 h or more than 8.5 h.

During 1 week before the study, subjects were asked to comply with standardized schedules (2300–0700 h bedtimes). Compliance was verified with wrist activity recordings (Actiwatch, Mini-Mitter Co.). In women, the study was initiated during the early follicular phase of the menstrual cycle.

The interventions (Figure 1) involved 3 days with 10-h bedtimes (2200–0800 h: B1–B3; baseline rested condition), followed by 8 days with 5-h bedtimes (R4–R11), with bedtimes always centered at 0300 h (0030–0530 h, circadian alignment) or with bedtimes delayed by 8.5 h on 4 days (0900–1400 h on days R5–R6 and R8–R9; circadian misalignment). Both interventions involved the same amount of bedtime restriction, representing 24 h of lost sleep opportunity over 8 days and were followed by 3 nights of recovery sleep.

An intravenous glucose tolerance test (IVGTT) was performed after an overnight fast at 0900 h on B2 and R10. Frequent blood sampling by an intravenous catheter was performed during B3 and R11. Levels of hsCRP were measured at 4-h intervals. Saliva samples for melatonin assays were obtained every 30 min, from 1600 h until bedtime, on R4 and R11.

Each participant met with a dietitian before the study to determine food preferences and select three nutritionally balanced menus that were served on a rotating basis. On blood sampling days (B3 and R11), identical carbohydrate-rich meals were served at 1400, 1900, and 0900 h and were completely ingested within 20 min. No other caloric intake was allowed on these 2 days. During the entire protocol, participants abstained from caffeinated beverages.

Sleep Data

Polygraphic sleep recordings (Neurofax EEG-1100A; Nihon Kohden, Foothill Ranch, CA) were scored visually at 30-s intervals in stages wake, I, II, slow-wave sleep (SWS) and rapid-eye-movement (REM) sleep, according to standardized criteria.[20] Total sleep time was defined as minutes of stages I + II + SWS + REM.


After three baseline samples, glucose (0.3 g/kg) was administered intravenously. Blood samples were taken at minutes 2, 3, 4, 5, 6, 8, 10, 12, 15, 19, 21, 22, 24, 26, 28, 30, 40, 50, 60, 70, 90, 100, 120, 140, 180, 210, and 240 after the glucose injection. At minute 19, insulin (0.02 units/kg) was administered intravenously. Minimal model analyses[21] were performed using the Minmod Millennium software[22] and provided SI, the acute insulin response to glucose (AIRg), a measure of β-cell response, and the disposition index (DI = AIRg × SI), a marker of diabetes risk.

One woman experienced hypoglycemia during her baseline IVGTT. Her IVGTT data were not included in the analysis.


Glucose concentrations were assayed at bedside (Model 23A; Yellow Springs Instrument Company, Yellow Springs, OH). Serum insulin and hsCRP concentrations were measured using IMMULITE high-sensitivity chemiluminescence assays (Diagnostic Products Corp.).

Melatonin was assayed in saliva and in serum by radioimmunoassay (Pharmasan Labs, Inc., Osceola, WI), with a limit of sensitivity of 3.5 pg/mL and an intra-assay coefficient of variation of 8%.

Circadian Phase

Circadian phase was determined by the "dim light melatonin onset" (DLMO) in saliva samples. Melatonin onset was defined as the first sample to exceed a threshold of 2 SD above the mean of the first three baseline samples (2000–2100 h) not followed by a return below this threshold. Light intensity was <50 lux at eye level. When the DLMO did not occur before bedtime, it was derived from serum levels for both study conditions. These estimations were made before the first (R4) and last (R11) short sleep periods.

hsCRP Levels

Seven determinations of hsCRP at 4-h intervals were obtained at baseline and at the end of sleep restriction. There were no consistent within-subject temporal variations, and therefore, we used the median of the seven values as a summary measure.

Statistical Analysis

Results are expressed as mean (SD) for normally distributed data or as median (25th, 75th percentile) otherwise. Data were log-transformed where applicable.

To examine the effect of sleep restriction within each group, cardiometabolic variables were submitted to repeated-measures ANOVA.

Because of well-documented sex differences in the regulation of sleep,[23,24] circadian rhythms,[25] and glucose metabolism,[26] sex was entered as a covariate in analyses comparing the two interventions. We examined the percentage change from baseline to the end of sleep restriction by using a factorial ANOVA with intervention, sex, BMI, and the interaction sex-by-intervention as factors for all cardiometabolic variables. All statistical calculations were performed using JMP software (SAS Institute Inc., Cary, NC).