The methods were the same as for our previous study (Kenya)24 except that the protocol had a delay of sleep in the last few days (11–13) rather than an advance.

Ethical Approval

The study was approved by the Rush University Medical Center Institutional Review Board and conformed to the standards set by the Declaration of Helsinki. Written informed consent was obtained from all subjects prior to their participation. Subjects were reimbursed for their participation.

Subjects

We enrolled 53 subjects 5 to 6 days before the start of the 14-day laboratory study. Of these, 47 started the study, 46 completed the study and 45 could be classified as African-American or European-American and are included in Table 1.

Subjects completed our Family/Ancestor Questionnaire and were asked to check all of the following categories that applied to them: White, Black or African-American, Asian, Hispanic or Latino, European, Middle Eastern, Far East Asian, Southeast Asian, Indian Subcontinent, North African, Afro-Caribbean, American Indian or Alaska native, Native Hawaiian or other Pacific Islander, Other, Don’t Know. A short description was provided for each category. Subjects did the same for their biological mother, biological father, and their four grandparents. All the European-Americans in Table 1 endorsed White for all 6 of their relatives except for one subject who checked “Don’t Know” for one grandparent. All of the African-Americans in Table 1 endorsed Black/African-American for all 6 of their relatives except for 6 subjects: 3 checked “Don’t Know” for two grandparents, one checked “Other” for one grandparent, one checked Indian Subcontinent for one grandparent and one checked “Afro-Caribbean” for father and two grandparents. None of the subjects checked “Hispanic or Latino” for any of their 6 relatives.

Buccal (cheek) swabs were used to collect a DNA sample from each subject during the study, and were processed by AncestrybyDNA, DNA Diagnostics Center, Fairfield, OH. This company performed biogeographical ancestry estimates based on ancestry informative markers, also known as population-specific alleles, which show large frequency differences between populations50,51. Results were returned several weeks later with percents for each subject in 4 categories: European, Sub-Saharan African, East Asian and Indigenous American (Table 1). The results section showed that there were weak, but statistically significant, correlations between ancestry (% European or % Sub-Saharan African) and circadian period and between ancestry and phase shift when all the subjects were combined, but when the groups were considered separately the correlations were very small and not significant. Thus, these ancestry estimates were not useful for predicting circadian variables within a race. The value in obtaining these DNA samples appears to be limited to making sure we categorized people into the correct groups. For example, in the current study one subject who completed the study listed both parents and all grandparents as being Black/African-American. The results of his genetic ancestry, however, indicated that he was 56% European and 30% Sub-Saharan African, so we could not include him in either the European-American or African-American group. A similar situation occurred for one self-identified African-American subject in the Kenya study24 who was 49% European and 46% Sub-Saharan African. We didn’t include their data because we want our results to generalize to the majority of African-Americans. This careful selection of subjects for the groups had not been done before our studies, and shows the benefit of confirming ancestry with DNA testing.

Subjects were young, mostly in their 20 s and 30 s, and healthy. They were not taking any prescription medications except for 6 women on oral contraceptives (3 African-Americans and 3 European-Americans). Due to the length of the study (14 days in the lab) most subjects were unemployed. Subjects were screened by telephone followed by an in-person interview and several questionnaires. Exclusion criteria included body mass index (BMI) >35 kg/m2, night shift work in the preceding month, smoking and excessive alcohol or caffeine consumption. Subjects were given urine tests for common drugs of abuse and nicotine, and were breathalyzed 5 to 6 days before starting the study and on days 1 and 7 of the study. Subjects completed the Munich Chronotype Questionnaire (MCTQ)52 and the Owl-Lark (Morningness-Eveningness) Questionnaire (MEQ)53 during the study (Table 1).

Protocol

This study took place in the Biological Rhythms Research Laboratory in Chicago from November 2014 to July 2016. Subjects were run in groups of three, and there was usually a mixture of African and European-Americans in each group.

During the first 5 days of the protocol (see Fig. 1), subjects were not given access to phones, lap tops, clocks, watches or any device that displays clock time. Their electronic items capable of time display were locked up from when they entered the lab on day 1 until after the phase assessment on day 6. During the ultradian LD cycles (LD 3:2) subjects lived in a large, windowless, room that contained 3 beds separated by partitions. There are overhead fluorescent fixtures on dimmers which were locked to the lowest level. Light intensities were measured frequently, at each subject’s eye, at the angle of gaze, with an Extech Model EA31 digital light meter. The research assistants took readings when subjects were in all the possible various positions in the room. Analysis of 1306 measurements showed that light levels were <50 lux 99% of the time, and <30 lux 84% of the time. The median light level was 18 lux. Subjects ate and drank ad lib, but were not permitted caffeine or alcohol. Showers (1/day) were at random times. Subjects were required to remain in bed during the dark periods even if they could not sleep and were monitored by an infrared camera. While awake they sat around a large round table and ate, played games, read, watched pre-recorded movies and TV shows or engaged in other sedentary activities. After the phase assessment on day 6, subjects napped in the dark from noon to 4 pm, and were then moved to the Bedroom Suite.

The Bedroom Suite has three bedrooms, a bathroom and a control room for research assistants. This was also a windowless environment and the bedroom and hallway lights were controlled by research assistants in the control room. Subjects had their own private bedrooms and were given their cell phones and any other electronics or watches they had brought with them (laptops, tablets, etc). Baseline sleep schedules (with 8 h time in bed, in the dark) were tailored to the individual using sleep diaries kept before entering the lab to best match the subject’s natural sleep time. Each bedroom had one overhead fluorescent ceiling fixture on a dimmer switch. Each subject’s bedroom fixture was set to its maximum for the first 10 h of the 16-h wake period, dimmed to the lowest level for the last 6 h, and turned off during the 8-h sleep episodes. Research assistants took readings when subjects were in various positions in the bedrooms and hallway. Analyses of 1214 measurements showed that during the high intensity hours light levels were most often between about 30 and 300 lux, and during the low intensity hours they were between about 5 and 60 lux. The median light level was 66 lux during the high intensity time, and it was 17 lux during the low intensity time.

Meals were served at scheduled times starting when subjects were woken from the second baseline sleep episode on day 8. Breakfast was 1 h after waking, lunch was 5 h after breakfast and dinner was 6 h after lunch. In addition, subjects were allowed 2 small snacks per day of <160 calories. Caffeinated beverages and alcohol were not permitted. Each bedroom had a large wall clock set to Chicago time. When the LD cycle and the sleep schedule were delayed 9 h, the time of meals was also delayed 9 h to keep meals in the same phase relationship to the sleep schedule. The clock on the wall in each subject’s room was changed to Japan time (9 h delay), and a sign underneath the clock was changed from “Chicago” to “Japan”. Each bedroom had a bulletin board where the subject’s times for bedtime, wake time, and meals were posted. The times on these signs did not have to change when the LD cycle was delayed, because the new times were in Japan time and matched the wall clock. As during baseline, the lights were set on high for the first 10 h of the wake period and on low for the last 6 h.

The dim light melatonin onset (DLMO), our measure of the circadian phase of the internal, master, circadian clock was measured during the phase assessments (Fig. 1). Saliva samples were collected every 30 min in very dim light (<5 lux) using Salivettes (Sarstedt, Newton, NC, USA). During the phase assessments subjects sat in La-Z-Boy recliners and usually watched pre-recorded movies and TV shows. Subjects were permitted to eat and drink ad lib, but no food or drink was permitted in the 10 min before each sample. Saliva samples were centrifuged, frozen, and later sent to SolidPhase, Inc. (Portland, Maine, USA) to be radioimmunoassayed (RIA) for melatonin. Each individual’s samples were analyzed in the same batch. The sensitivity (limit of detection) of the assay was 0.9 pg/ml. Intra-assay coefficients of variation for low (daytime), medium (evening) and high (nighttime) levels were 20.1, 4.1 and 4.8% respectively. The inter-assay coefficients of variation for low, medium and high levels were 16.7, 6.6 and 8.4%, respectively.

Buccal (cheek) swabs for DNA were taken after subjects were awakened from the first baseline sleep episode on day 7, before they drank or ate anything. One hour after their wake up time they were allowed to leave the lab and go outside for 8 h. That was the only time during the entire 14-day study that they were not constantly supervised by research assistants 24 h a day. When they returned from this 8-h break they were given a urine drug screen to test for common drugs of abuse and nicotine, and were breathalyzed for alcohol. All subjects passed these screens.

Data Analysis

Melatonin profiles were smoothed with a locally weighted least squares (LOWESS) curve set to medium, 10 points in the smoothing window (GraphPad Prism, GraphPad Software Inc., La Jolla, CA, USA). The threshold for determining the DLMO was 25% of the distance from the fitted minimum value to the fitted maximum value, i.e. minimum +25% (maximum − minimum). The second and fourth phase assessments (the two final assessments) were longer than the first and third (baseline) (Fig. 1). Therefore, the threshold from the final in each baseline/final pair was used to determine the DLMOs for that pair.

To calculate the free-running circadian period (τ) the difference between the DLMOs on the first and second phase assessment (Fig. 1) was divided by 4 (because there were 4 days between these DLMOs) and then added to 24 (when the DLMO delayed), or subtracted from 24 (when the DLMO advanced). For example if the DLMO on the first assessment was 21:00 and the DLMO on the second was 22:00, then the difference is 1.0 h. We divide this difference by 4 which equals 0.25 h. The circadian period is thus 24.25 h. If the DLMO on the first assessment was 23:00 and the DLMO on the second was 22:00, then the circadian period would be 23.75 h.

To calculate the phase shift of the circadian clock due to the 9-h delay of zeitgebers the DLMO on the fourth phase assessment was subtracted from the DLMO on the third phase assessment (Fig. 1). Thus, if the DLMO on the third assessment was 21:00 and the DLMO on the fourth was 23:00 then the phase shift would be −2.0 h. By convention delays are indicated with a negative number.

We used t-tests to examine differences between African-Americans and European- Americans in variables such as the free-running period and phase shift, and for a sex difference in circadian period among the European-Americans. Pearson correlation coefficients were used to test for associations among the main outcomes measures. All reported tests are based on 2-tailed probabilities. Results are presented as means ± SD unless otherwise indicated. GraphPad Prism was used for data analysis.