Influence of Nocturnal Light Exposure on the Impairment of Glucose Tolerance Induced by Chronic Sleep Restriction
Phase: NA
Conditions: Glucose Intolerance, Sleep Deprivation, Sleep
Sponsor: Brigham and Women's Hospital

Inﬂuence of Nocturnal Light Exposure on the Impairment of Glucose Tolerance 
Induced by Chronic Sleep Restriction 
 
NCT04868539 
 
Last modiﬁed: 11/26/2024 
 

 
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2021-P-000961 Detailed Protocol 
 
I. 
BACKGROUND AND SIGNIFICANCE 
This project is designed to test for the first time whether glucose metabolism is differentially impaired by sleep 
restriction with and without additional exposure to artificial light at night (ALAN). Laboratory studies have shown 
that sleep restriction to 4-6h per night for durations varying from one to 14 days reduces glucose tolerance in 
otherwise healthy adults [10-23,32,33], but the mechanisms by which insufficient sleep impairs glucose 
metabolism are still unknown. Current theories are based on the premise that the adverse metabolic 
consequences are caused by reduction in the duration of sleep per se [34]. However, sleep curtailment is typically 
accompanied by longer exposure to artificial light at night (ALAN), which is an environmental endocrine disrupter 
that profoundly disrupts circadian rhythms [24-27,35-37].  
 
We have previously reported that acute circadian misalignment induced hyperglycemia comparable to pre-
diabetic states in a third of otherwise healthy participants [55]. Since then, we have shown that even when the 
circadian phase of participants was realigned, prior exposure to 2 ½  weeks of chronic sleep restriction combined 
with a history of recurrent circadian disruption induced even more deleterious effects on glucose metabolism 
[32], in which pancreatic beta cells failed to respond adequately to increased glucose levels. Moreover, both 
night and rotating shift work (which induce circadian disruption) are associated with increased risk for metabolic 
problems (81)(82)(83)(84)(85)(86)(87). Night shifts can lead to acute increases in glucose and insulin levels (88), 
although some studies report reduced insulin release in response to meals consumed during the night (89-91). 
Given that circadian disruption has been shown to independently adversely affect metabolism, and exposure to 
ALAN adversely impacts metabolism in animals [38], it is important to understand the extent to which circadian 
disruption contributes to the observed impact of sleep curtailment on metabolism[35]. No previous studies of 
the metabolic impact of sleep restriction in humans have controlled for this additional exposure to ALAN, 
thus confounding the effects of sleep restriction with the effects of circadian disruption caused by 
extended exposure to ALAN. 
 
Seminal work by Spiegel and colleagues [10] demonstrated that 6 nights of sleep restriction to 4h per night 
caused impairments in glucose tolerance in otherwise healthy young men. Subsequent sleep restriction studies 
have replicated and expanded on these initial results [11-23,32], establishing a clear association between sleep 
restriction and disrupted glucose tolerance. Many of these studies employed rigorous methods for ensuring the 
participants remained awake throughout the sleep restriction [13-15,19,22,32], for recording sleep [13-17,19,32], 
and for assessing metabolism [11,13,14,16,17,19,20,22,32], although this was not true for all. In fact, in some 
cases participants were only provided test meals (and otherwise had uncontrolled diets [21]), participants were 
allowed to leave the laboratory during the day (so that wakefulness and adherence to the study diet were not 
controlled [10,16,21]), baseline sleep was scheduled at fixed times that may have induced difficulty sleeping [14-
16], or objective evidence for adherence to the extended wake episodes was not documented (potentially 
missing the presence of brief naps in sleep-restricted individuals).  
 
Importantly, none of the previous sleep restriction studies have controlled for longer exposure to ALAN when 
imposing an extended wake duration [10,11,13,15,16,20-22]. Individuals with restricted sleep, whether in the 
laboratory (31, 130, 131) or in normal life (65), are exposed to extended photoperiod and to altered meal timing, 
which can differentially entrain central and peripheral circadian oscillators (98, 126). Retinal exposure to ordinary 
indoor light can impact the human circadian system (132-137), and during sleep restriction additional light 
exposure occurs in the late evening and/or early morning, times when the circadian system is most sensitive to 
light (137-142). This additional nighttime light exposure may cause phase shifts (143) and melatonin suppression 
(136, 137). However, most sleep restriction studies have not assessed or controlled for such circadian 
disruptions. There is also evidence from animal studies that, in addition to light exposure, sleep loss can have a 
direct impact on neuronal activity in the master circadian pacemaker (144), which in turn can influence peripheral 
clocks in metabolic organs through neuronal and hormonal signals [reviewed in (43, 101)]. Thus, given that 
exposure to ALAN is known to be an endocrine disruptor and to profoundly disrupt circadian rhythms, and 
endocrine and circadian disruption can adversely affect metabolism [28,29], it is probable that endocrine and 
circadian disruption caused by the extended exposure to ALAN may contribute to the adverse metabolic effects 

 
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induced by chronic sleep restriction. We have therefore designed this study to be a direct comparison 
between sleep restriction with and without extended exposure to ALAN in order to distinguish the impact 
of sleep loss per se and sleep loss with extended photoperiod on glucose metabolism.  
 
Determining whether endocrine and circadian disruption induced by ALAN contributes to the 
mechanism by which chronic sleep deficiency leads to the development of obesity, insulin resistance, 
and type 2 diabetes is important given the widespread prevalence of sleep deficiency in Americans of 
all ages. By clarifying a potential mechanism by which sleep restriction impairs glucose 
metabolism, our findings will lay the groundwork for the development of novel treatments and 
countermeasures to mitigate the adverse metabolic effects of chronic sleep restriction. If 
extended ALAN-induced endocrine and circadian disruption is one of the mechanisms by which sleep 
restriction impairs glucose metabolism, targeted countermeasures could be developed (e.g. eyeglasses 
that minimize the endocrine and circadian disruptions induced by ALAN by reducing exposure to short-
wavelength artificial light, use of lamps with a lower color temperature to minimize exposure to short-
wavelength artificial light during episodes of extended wakefulness, software to reduce exposure to 
short-wavelength light from light-emitting screens, etc).  
 
II. 
SPECIFIC AIMS 
The goal of this project is to evaluate whether extended duration ALAN contributes to the adverse effects of 
sleep restriction on glucose metabolism. We plan to use a crossover design consisting of a 7-day baseline, 7-
day sleep restriction (to 5h per night) with (LD 19:5) or without (LD 14:10) ALAN, 9-day washout, and another 7-
day sleep restriction with or without ALAN (Figure 8). 20 healthy participants will be randomly assigned to receive 
ALAN in either the first or second exposure, balanced by age and sex. Specifically, we plan to: 
1. Test the hypothesis that exposure to one week of sleep restriction with concurrent exposure to extended 
duration ALAN (LD 19:5) will induce greater impairment of insulin sensitivity than exposure to one week 
of sleep restriction without extended duration ALAN (LD 14:10). Insulin sensitivity (SI) will be assessed 
via an intravenous glucose tolerance test (ivGTT) before and after each week of exposure. 
2.  Test the hypothesis that exposure to one week of sleep restriction with concurrent exposure to extended 
duration ALAN (LD 19:5) will induce greater impairment of glucose tolerance than exposure to one week 
of sleep restriction without extended duration ALAN (LD 14:10). The 90-minute glucose area under the 
curve (AUC) will be assessed in response to a standardized meal both before and after each week of 
exposure.  
3. Test the hypothesis that exposure to one week of sleep restriction with concurrent exposure to extended 
duration ALAN (LD 19:5) will reduce the duration of nocturnal melatonin secretion as compared to 
baseline more than exposure to one week of sleep restriction without extended duration ALAN (LD 
14:10). The 24-h profile of melatonin secretion will be assessed during the lighting conditions that prevail 
throughout both sleep restriction conditions (i.e., 5h in darkness per night vs. 10h in darkness/near 
darkness per night). To determine the mechanism of this anticipated decrease in the duration of melatonin 
secretion (acute light-induced melatonin suppression vs. a reduction in the duration of the endogenous 
melatonin secretory profile), we will also measure the endogenous melatonin secretory profile on a ~24-
hr recording on the day immediately after each week of intervention.  
4. Exploratory Aim 4: Test the hypothesis that exposure to one week of sleep restriction with concurrent 
exposure to extended duration ALAN (LD 19:5) will lead to a greater reduction in the levels of long-chain 
highly unsaturated triglycerides than exposure to one week of sleep restriction without extended duration 
ALAN (LD 14:10). Plasma lipids will be assessed via mass spectrometry for a baseline and sleep-
restricted sample from each participant in the above studies under ALAN and non-ALAN conditions. 
Because lower levels of long-chain highly unsaturated triglycerides are associated with a loss of insulin 
sensitivity [30], we will assess whether sleep restriction with ALAN leads to adverse changes in the levels 
of these triglyceride species that do not occur when sleep restriction is conducted without ALAN. 
5. Exploratory Aim 5: test the hypothesis that exposure to one week of sleep restriction with concurrent 
exposure to extended duration ALAN (LD 19:5) will lead to a difference in the exhaled VOC profile 
compared with exposure to one week of sleep restriction without extended duration ALAN (LD 14:10).  

 
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6. Exploratory Aim 6: test the hypothesis that exposure to one week of sleep restriction with concurrent 
exposure to extended duration ALAN (LD 19:5) will lead to a change in the proteomic profile compared 
with exposure to one week of sleep restriction without extended duration ALAN (LD 14:10).  
7. Exploratory Aim 7: test the hypothesis that the BodyTime assay will track changes in the melatonin profile 
and show a change in timing after exposure to one week of sleep restriction with concurrent exposure to 
extended duration ALAN (LD 19:5) compared to baseline.  
8. Exploratory Aim 8: test the hypothesis that exposure to one week of sleep restriction will induce changes 
in objective human vocal parameters, both with and without extended duration ALAN.  
 
III. 
SUBJECT SELECTION 
A total of 20 healthy adults (20-40 years old) will be studied in this proposal. In order to obtain 20 people who 
meet all the study criteria and complete the 33-day inpatient study, we expect to enroll up to 100 people in the 
screening process for the study. Based on similar studies in the past, we anticipate that up to 25 individuals 
may be enrolled in the main study in order to have 20 complete the study. Approximately equal numbers of 
men and women will complete the study in each group.  
 
Each potential participant will undergo an extensive screening procedure prior to participation in the study. The 
procedure begins with a telephone/online screening (to determine general medical suitability) and answering 
any questions potential participants may have about the study. If the original preliminary telephone/online 
screening questionnaire reveals no grounds for exclusion (see below), then potential participants are asked to 
come for an interview and physical screening. The physical screening includes a medical history, blood and 
urine tests, an ECG, and a physical examination by a physician or other qualified personnel (i.e physician 
assistant, certified nurse practitioner). Participants will also undergo psychological screening (see below), 
including questionnaires and a structured interview with a clinical psychologist. Finally, participants will be 
screened for sleep apnea and periodic limb movement disorder using a home sleep test.   
 
We estimate that up to 10 individuals will inquire about the study for every 1 eventually studied, and that up to 
5 individuals will begin the screening process for every 1 who successfully completes the screening and begins 
the study. We therefore plan to enroll up to 100 participants in the study in order to have 20 who pass all the 
screening and complete the 33-day study. 
 
Inclusion/exclusion criteria.  
(a) Medication/drug use. Potential participants must report no history of drug or alcohol dependency. Potential 
participants must report moderate or no use of caffeine, cigarettes and alcohol by history; be willing to abstain 
from caffeine, nicotine, alcohol and herbal medications for the duration of the ambulatory segment of the study; 
and will not be allowed to use these substances during the laboratory segment of the study. Potential participants 
who are regularly taking any prescription (with the exception for hormonal birth control; see Inclusion of Women 
and Minorities section) or over-the-counter medication will be excluded.  
 
(b) Evaluation of medical suitability. Potential participants must be ambulatory and have no major visual or 
auditory handicaps. Participants must be free from any acute or debilitating medical conditions. Medical suitability 
will be determined by clinical history, physical examination, electrocardiogram, clinical biochemical screening 
tests of blood and urine.  
 
Any participant with symptoms of acute or active illness (e.g., fever, leukocytosis, hypertension) will be excluded 
from study until those symptoms resolve. Given the wide range of illnesses that are encountered in medical 
practice, it would not be possible to provide a comprehensive list of each and every disease that could serve as 
grounds for exclusion for the participant. However, the following summarizes common medical conditions that 
will be exclusionary.  
 
Diseases of the Cardiovascular System: hypertension (systolic blood pressure >130 or diastolic blood pressure 
>80), heart failure, cardiomyopathy, cor pulmonale, ischemic heart disease, valvular heart disease, history of 
heart transplantation, cardiac tumors, pericardial disease;  
 

 
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Disorders of the Respiratory System: asthma, cystic fibroses, chronic bronchitis, emphysema and airway 
obstruction, interstitial lung diseases, pulmonary hypertension, lung neoplasms, ARDS;  
 
Disorders of the Kidney and Urinary Tract: acute or chronic renal failure, history of renal transplantation, 
tubulointerstitial diseases of the kidney, urinary tract obstruction, tumors of the urinary tract;  
 
Infectious Diseases: infective endocarditis, HIV infection, sexually transmitted diseases [e.g., syphilis (including 
congenital syphilis and its sequelae), gonorrhea], osteomyelitis, brucellosis, tuberculosis, leptospirosis, Lyme 
disease, mononucleosis, parasitic infections such as toxoplasmosis, giardiasis, schistosomiasis, leishmaniasis;  
 
Disorders of the Gastrointestinal System: esophagitis, peptic ulcer, gastritis, neoplasms of the esophagus, 
stomach or bowel, disorders of absorption, inflammatory bowel disease, diseases of the small and large intestine, 
acute appendicitis, hepatitis, cirrhosis or neoplasms of the liver, history of liver transplantation, diseases of the 
gallbladder and bile ducts, pancreatic disease, diabetes;  
 
Disorders of the Immune System, Connective Tissue and Joints: AIDS, systemic lupus erythematosus, 
rheumatoid arthritis, scleroderma, ankylosing spondylitis, vasculitis, sarcoidosis, fibromyalgia;  
 
Disorders of the Hematopoietic System: anemia, leukemia, myeloproliferative diseases, history of bone marrow 
transplantation;  
 
Neoplastic Diseases: lymphoma, carcinoma, melanoma, or any other neoplastic diseases;  
 
Endocrine and Metabolic Diseases: thyroid disease, Addison's Disease, Cushing's Syndrome, aldosteronism, 
hypoaldosteronism, pheochromocytoma, diabetes, disorders of sexual differentiation, disorders of 
neuroendocrine regulation, diseases of the anterior pituitary and hypothalamus, hemochromatosis porphyria, 
Wilson's Disease, glycogen storage diseases, diseases of the parathyroid gland, metabolic bone disease, 
disorders of phosphorus or magnesium metabolism, Paget's Disease;  
 
Neurologic Disorders: epilepsy and disorders of consciousness, dementia, amnesic disorders, neoplastic 
diseases of the central nervous system, demyelinating diseases, Parkinson's Disease, muscular dystrophy, 
myasthenia gravis, periodic paralysis, dermatomyositis, polymyositis, infections of the nervous system, stroke, 
history of transient ischemic attacks, hydrocephalus, tumors of the pituitary gland, pinealoma, intervertebral disc 
disease, ataxia, Gilles de la Tourette Syndrome, Huntington's Disease, tardive dyskinesia, history of recurrent 
migraine or cluster headaches, neuromuscular disease.  
 
Sleep Disorders: narcolepsy, sleep apnea, Periodic Limb Movement Disorder (PLMD), nocturnal paroxysmal 
dystonia, REM-sleep behavior disorder, nocturnal eneuresis.  
 
(c) BMI. BMI inclusion criteria are 20 – 29.9 kg/m2.  
 
(d) Evaluation of psychiatric/psychological suitability. Under the supervision of Dr. 
 
 Ph.D., 
Associate Psychologist in the Division of Psychiatry at the Brigham and Women's Hospital/Harvard Medical 
School, each potential participant will be administered an MMPI II, Symptom Checklist 90R, Beck Depression 
Scale, and State Anxiety Scale. Dr. 
 is a clinical psychologist who has been carrying out psychological 
screening for participants in our research studies for more than three decades, and support for these pre-study 
evaluations is included in the proposed project budget. As the final part of the psychological screening evaluation 
of potential participants, Dr. 
 will conduct a structured interview (SCID-R) with each potential participant. 
In particular, Dr. 
 will evaluate potential participants for Axis II personality types that might interfere with 
compliance or with their personal ability to tolerate the conditions of the study. Dr. 
 will also evaluate 
whether potential participants demonstrate a full understanding of the requirements and demands of the study.  
 
Individuals with a personal history of psychiatric illnesses or psychiatric disorders will be excluded, and those 
with a first-degree relative with psychiatric illness will also be excluded. That includes alcoholism, drug 

 
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dependency, major mood disorders such as major depression and manic depressive illness, schizophrenic 
disorders, anxiety disorders including panic disorder, generalized anxiety disorder, post-traumatic stress 
disorder, obsessive compulsive disorder, agoraphobia, claustrophobia, paranoid personality disorder, schizoid 
personality disorder, schizotypal personality disorder, borderline personality disorder, and antisocial personality 
disorder. Individuals who are unaware of specific psychiatric diagnoses but who have a history of having been 
treated with antidepressant, neuroleptic medications, or major tranquilizers will be excluded from study. 
However, a personal history of limited prior counseling or psychotherapy (e.g., for adjustment reactions) will not 
necessarily be exclusionary. Participants must demonstrate a full understanding of the requirements and 
demands of the study.  
 
=============================================================  
Exclusionary Scores on Psychological Screening Tests  
1) MMPI Scales:  
Depression scale  
 
 
 
 
T > 70: excluded automatically  
Psychopathic deviance, schizophrenia,  
 
T > 75: excluded automatically*  
hypomania scale  
L, F & K validity scales & other clinical scales  
T > 80: excluded automatically  
L, F & K validity scales & other clinical scales  
T >70, but <80: held pending interview**  
2) Beck Depression Scale  
 
 
 
> 10: excluded automatically  
3) State Anxiety Scale 
 
 
 
> 40: excluded automatically  
4) Symptom Checklist 90-R  
Depression scale Distress level  
 
> 1.25: excluded automatically  
Hostility scale   
 
 
 
> 1: excluded automatically  
Phobic anxiety  
 
 
 
> 0.75: excluded automatically  
Paranoid ideation  
 
 
 
> 1.25: excluded automatically  
Psychoticism   
 
 
 
> 1: excluded automatically  
Anxiety 
 
 
 
 
> 1.25: excluded automatically  
 
*Scores on all 3 of these scales are correlated with age; college and graduate students generally score higher 
on these scales, so we have adjusted our cutoff values accordingly.  
 
**Participants with these scores may be excluded; they will be referred to Dr. 
 for clinical interview, and 
inclusion/exclusion will depend on the results of that clinical interview.  
=============================================================  
 
(e) Work, travel, and sleep-wake history. Participants must report habitually sleeping between 7 and 9 hours 
per night. This will be verified using actigraphy and sleep diaries during the pre-study Ambulatory Baseline. 
Potential participants will complete the Pittsburgh Sleep Quality Index and the Epworth Sleepiness Scale prior 
to study, and individuals with evidence of significant sleep disruption (PSQI >5 or ESS >10) will be excluded. 
Individuals with a prior diagnosis of a sleep disorder, and those regularly taking prescription or over-the-counter 
sleep aids will be excluded.  
 
Potential participants must have no history of working on an irregular schedule that requires overnight, early 
morning (shift starts before 6am), and daytime work for the three years prior to study. They must not have 
traveled across more than two time zones during the month prior to the study. These criteria are designed to 
ensure that the individual is stably entrained to local environmental time and to their individual sleep-wake 
schedule.  
 
For the two weeks prior to entering the laboratory for study, all participants will maintain a regular sleep-wake 
and light-dark schedule (± 30 min) of 9-h duration, centered at their habitual times, will record these times in a 
sleep diary, and will phone in twice per day to record their sleep and wake times. They will also undergo 
ambulatory monitoring of their activity and light exposure levels using a small wrist activity monitor during those 
same two weeks to document their sleep-wake behavior prior to entering the laboratory, and to ensure they enter 
the study in a well-rested condition.  

 
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Inclusion of racial/ethnic minorities. Our recruitment procedures provide all applicants with an equal 
opportunity to participate in our studies regardless of race, ethnicity, or national origin.  
 
Gender. We will attempt to include equal numbers of women and men in the proposed study. However, pregnant 
subjects or those attempting to become pregnant will not be studied. Women with symptoms of pre-menstrual 
dysphoric disorder (PMDD) will be excluded, as will those who are breastfeeding. Women who have been 
pregnant or who have been breastfeeding within the past 6 months will be excluded. 
 
Source of subjects and recruitment methods.  
 
We will recruit participants from the local community by posting ads on the internet and posting flyers on local 
bulletin boards, methods we find to be cost-effective for this target population. We will also use two methods 
within Partners Health Care,  Research Patient Data Registry (RPDR)and Rally RPDR is a searchable database 
maintained by MGB hospital systems to provide patients that meet user-defined characteristics and criteria such 
as diagnoses, procedures, medications, and/ or laboratory results.  We will also recruit previous study 
participants from a database of former participants who have given permission to be notified about future studies 
that they might be eligible for. Research Invitations will be used for direct recruitment of any eligible subjects who 
have not opted out of receiving Research Invitations and patients who opted out will be filtered out of the 
recruitment list and they will not be sent the Research Invitation. The PI will conduct ongoing monitoring of patient 
responses to ensure that our selection criteria are identifying the right patients. Complaints about this method of 
recruitment will be submitted to the IRB as an Other Event.  The identity of potential volunteers is maintained 
until 
they 
choose to 
contact the Investigators. 
Rally 
is 
the 
Partners 
clinical 
trials 
website 
(clinicaltrials.partners.org) where Investigators can post study advertisements and interested community 
members can view all studies that match their age, sex, and other criteria. We will also recruit previous study 
participants. Drs. Czeisler, Duffy, Scheer, and Williams each carry out clinical research on healthy adults, and 
participants are asked at the end of their study if they wish to be notified about future studies that they might be 
eligible for.  
 
IV. 
SUBJECT ENROLLMENT 
This study uses a within-subjects randomized crossover design to compare the effects of sleep restriction with 
and without ALAN on glucose metabolism. The order in which subjects undergo the two sleep interventions will 
be randomized.   
 
Procedures for obtaining informed consent (including timing of consent process) 
Written informed consent will be obtained from each subject in a 2-step process. A Screening Consent Form will 
be used to obtain consent for the screening procedures. This will be obtained by a Study Coordinator/Recruiter 
or post-doctoral research fellow at the start of the first screening visit. A Research Consent Form will be used to 
obtain consent for the research study itself. Consent for the screening procedures will be obtained by a trained 
Research Assistant. Consent for the research study procedures will be obtained by the PI, a Co-Investigator, or 
a Research Fellow at least 24h before the first study day. We plan to use a REDCap eConsent module and 
secure videoconferencing (MGB-approved and HIPPA-compliant Zoom) or to do the consent in person when 
public health guidelines allow. 
 
Participants who complete the prescreening form have the option to consent to being contacted by text message 
for study visit reminders during the screening process only. The MGB recommended consent text disclosing 
security risks of text messaging is shown to participants at the end of the prescreening form, and participants 
have the option to opt in to receive text reminders. Text reminders are not used during the inpatient study, nor 
are they used after completion of the research study. Text messaging will be carried out using Twilio, a secure 
texting platform integrated with REDCap through the REDCap + Twilio Module.    
 
 
V. 
STUDY PROCEDURES 
The experimental protocol consists of 6 segments: ambulatory baseline; laboratory baseline assessment of 
glucose metabolism and circadian rhythms; first sleep intervention including re-assessment of glucose 

 
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metabolism and circadian rhythms; washout followed by second baseline assessment of glucose metabolism 
and circadian rhythms; second sleep intervention including re-assessment of glucose metabolism and circadian 
rhythms, and recovery. The ambulatory baseline is roughly 2 weeks, after which participants will be studied in 
the laboratory for a 33-day protocol.  
 
During the Ambulatory Baseline, wrist activity and light levels will be recorded in participants at home for 
approximately 2 weeks prior to study admission. They will be asked to wear an activity monitor (MotionWatch 8 
or similar, CamNTech, Inc.) on the wrist of the non-dominant hand, maintain a regular self-selected sleep-wake 
schedule with 9h time in bed each night, record this schedule in a sleep diary, and call in to our time-stamped 
call-in telephone system just prior to each bedtime and immediately after each wake time. These procedures will 
ensure the participants are stably entrained to their schedule and well-rested when the study begins. We may 
also ask them to log the time, content, and amount of food and drink consumed each day during this time in a 
food diary. This will inform us of the participant’s usual diet and allow comparison with the laboratory diet. 
 
Immediately after the ambulatory baseline, the participant will be admitted to the Center for Clinical Investigation 
(CCI) at BWH on Day 1. They will be scheduled to sleep for 10 hours overnight at the CCI (centered at their 
usual times), and their sleep will be recorded with standard polysomnography (PSG) on some or all of the nights. 
Upon awakening the next morning (Day 2), the participant will begin the first Baseline Day. They will be given 
isocaloric, nutrient-controlled meals (breakfast, lunch, dinner) and will be scheduled to sleep again for 10 hours 
overnight. This will continue for the remaining baseline days. Near the end of the first Baseline segment 
(approximately Day 5), they will have an IV catheter inserted into a forearm vein for blood sampling. After 
awakening the next morning (~Day 6) after an overnight fast, the participant will undergo an assessment of their 
resting metabolic rate via indirect calorimetry. This will be followed by a Mixed Meal Response at breakfast to 
evaluate their baseline glucose and insulin response to a meal (see below for details). Frequent blood samples 
for melatonin will also be drawn to assess the duration of melatonin secretion and melatonin levels during the 
metabolic assessments, and this sample collection will continue throughout the next three days (~Days 6-8). 
Upon awakening on Day 7 after an overnight fast, the participant will undergo an assessment of their resting 
metabolic rate via indirect calorimetry again, and this will be followed by a baseline insulin-modified IV glucose 
tolerance test (GTT) that will be carried out to assess insulin sensitivity (SI), acute insulin response (AIRg), and 
glucose effectiveness (see below). The IVGTT will be scheduled to begin at the same time as the Mixed Meal 
Response on the previous day. Sample collection for the IVGTT will be completed by approximately 3 hours later 
(4 hours after wake time). Beginning in the early afternoon, ambient lighting will be lowered to <15 lux and ~hourly 
blood samples for melatonin endogenous circadian rhythm assessment will be drawn. Sleep will be scheduled 
at the same time as Nights 1-6, and blood sampling will continue throughout the night. Sample collection for the 
circadian rhythm of melatonin will be completed by approximately 10 hours after wake time, at which time the 
sampling IV will be removed and the lighting will be returned to normal.  
 
The next 7 nights are the First Sleep Intervention and will depend on the randomization order. In the Sleep 
Restriction Group with Extended Duration Artificial Light At Night (ALAN) first, the sleep episodes will be 
shortened to 5 hours, centered at the same time as the baseline sleep (with bedtime 2.5 hours later and wake 
time 2.5 hours earlier). In the Sleep Restriction group without Extended Duration ALAN, the sleep episodes will 
be shortened to 5 hours as in the ALAN Condition (centered at the same time as the baseline sleep with bedtime 
2.5 hours later and wake time 2.5 hours earlier), but the participant will remain sitting in bed in near darkness (< 
1 lux) for the 2.5 hours before and after the 5-hour sleep episode, such that their exposure to room lighting and 
activity (14 hours/day) will remain similar to that in the Baseline condition. The timing of meals in both conditions 
throughout this intervention segment will remain the same as on the Baseline days.  
 
Near the end of the first sleep intervention (~Day 14), each participant will undergo a re-assessment of their 
resting metabolic rate via indirect calorimetry at the same time as during Baseline. This will be followed by a 
Standardized Mixed Meal Response at breakfast to re-evaluate their glucose and insulin response to a meal, 
along with frequent blood samples to assess duration of melatonin secretion and melatonin levels during the 
metabolic assessments. This sample collection for melatonin will continue throughout the next three days (~Days 
14-16). Near the end of the first sleep intervention (~Day 15), an ivGTT re-assessment will be carried out to 
assess insulin sensitivity (SI), acute insulin response (AIRg), and glucose effectiveness. This will begin at the 

 
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same time as the Baseline ivGTT. Sample collection for the ivGTT will be completed by approximately 3 hours 
later. Beginning in the afternoon, ambient lighting will be lowered to <15 lux and frequent blood samples for re-
assessment of melatonin and cortisol circadian rhythms will be drawn, continuing until the following afternoon . 
   
For the Washout, sleep episodes will be scheduled to 10 hours each night at the same time of the Baseline 
sleep episodes, from Days 15-23. Upon awakening after an overnight fast toward the end of the Washout (~Day 
23), the participant will undergo the second Baseline assessment of their resting metabolic rate, glucose and 
insulin responses, and melatonin rhythms. Standardized Mixed Meal Responses, ivGTTs, and melatonin 
sampling will be carried out on the next 3 days (~Days 23-25) as performed during the first Baseline assessment 
on Days 6-8. Following the washout, the next 7 nights are the Second Sleep Intervention, during which 
participants will undergo the sleep restriction condition (with or without ALAN) which they have not yet undergone 
during the first sleep intervention.  
 
Upon awakening after an overnight fast at the end of the 7-day exposure (~Day 31), the participant will undergo 
another re-assessment of their resting metabolic rate, glucose and insulin responses, and melatonin rhythms. 
Standardized Mixed Meal Responses, ivGTTs, and melatonin sampling will be carried out on the next 3 days 
(~Days 31-33) as performed during the assessment following the first Sleep Intervention on Days 14-16. The 
final Recovery sleep episode on Night 32 will be scheduled for 10 hours at the same time of the Baseline sleep 
episodes. Blood sampling will be completed approximately 10 hours after waketime on Day 33, at which time 
the sampling IV will be removed and the participant will be discharged. At the times indicated above when blood 
sampling is scheduled, we may collect additional small amounts of blood to assay for proteins, lipids, transcripts, 
or metabolites. 
 
How Biospecimens, Data, and/or Records, will be Obtained. As outlined above, activity/light data and diary 
recordings, as well as a blood sample for DNA extraction will be collected from participants during the pre-study 
screening period. In the laboratory study, PSG sleep and wake recordings, answers to written and computerized 
questionnaires, whole blood and saliva biospecimen will be collected. We may also collect breath samples and 
fecal samples during specific days of the study. 
 
Ambulatory physiologic monitoring. Wrist activity and ambient light levels will be monitored in participants at 
home for approximately 2 weeks prior to study admission. They will be asked to wear an activity monitor 
(MotionWatch 8 or similar, CamNTech, Inc.) on the wrist of the non-dominant hand, maintain a regular self-
selected sleep-wake schedule with 10 h time in bed each night, record this schedule in a sleep diary, and call in 
to our time-stamped call-in telephone system just prior to each bedtime and immediately after each wake time. 
These procedures will ensure the participants are stably entrained to their schedule and well-rested when the 
study begins. They will also log the time, content, and amount of food and drink consumed each day during this 
time in a food diary. This will inform us of the participant’s usual diet and allow comparison with the laboratory 
diet. 
 
Inpatient environment and conditions. Participants will live in environmentally-controlled private rooms in the 
Intensive Physiological Monitoring (IPM) Unit of the Brigham and Women’s Hospital (BWH) Center for Clinical 
Investigation (CCI) throughout the study. The rooms are equipped with hand-held terminals for on-line event 
recording, a porthole to enable 24-h blood sample collection without sleep disturbance, a closed-circuit camera 
and a voice-activated microphone for continuous monitoring, and neurobehavioral performance testing computer 
stations. Technicians are present 24h/ day to carry out the protocol, monitor the data acquisition systems, collect 
biologic specimen, record PSG, and respond to participant’s requests. Research nurses are present 24h/day to 
insert and maintain the intravenous blood sampling system and to monitor the participant’s health and mood, 
and an attending physician visits the participant daily to monitor their health. Specially-trained nurses, PAs and 
NPs are available to carry out IVGTTs. A dedicated specimen processing staff are present 24h/day to ensure all 
specimen are processed according to the requirements for the specific analytes to be assayed. Written protocols 
and checklists are used to ensure uniformity in the execution of standard procedures and to foster intra-staff 
communications (e.g., at shift change). Our research group has studied participants using these conditions for 
more than 30 years, and the staff members of the Division and the BWH CCI carry out such studies in the IPM 
on a daily basis. 

 
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Wake episodes. During this condition, on most days the participants will be free to move about their study suite 
as desired, except that they are instructed not to lie down or nap. The participant's activity will be monitored for 
compliance by means of closed-circuit cameras, and during the intervention week a study team member will 
remain in the room with the participant to ensure they do not fall asleep accidentally. See Sleep restriction section 
below for further details.  
 
Lighting. During the circadian rhythm sample collection segments on Days 7-8, 15-16, and 24-25, maximum 
ambient light during scheduled wake will be <15 lux when measured vertically at 72" [90 deg] and ~5 lux 
measured horizontally at a height of 54" [0 deg]; “dim lighting”). In the Sleep Restriction without Extended 
Duration ALAN condition, the participants will be in “near darkness” for the 2.5 hours before and after each 
scheduled 5-hour sleep episode. This lighting will approximately 3 lux maximal exposure, measured at 72" 
vertically or <1 lux measured at 54" horizontally). Throughout all other wake episodes “normal indoor lighting” 
will apply. Ambient lighting is switched off (0 lux) during all scheduled bed rest episodes. 
 
Sleep and polysomnographic recording. Throughout all scheduled sleep opportunities, participants will be 
required to remain supine in bed in the dark. Sleep episodes will be polysomnograhically recorded on some or 
all nights to document the duration of sleep [108] using Vitaport-3 ambulatory digital sleep recorders (DSR) or 
similar devices. Study staff will apply surface electrodes to specific face and scalp locations allowing sufficient 
time for the participant to dress and get into bed, and for the technical staff to connect the DSR and carry out 
calibration procedures. EEG will also be recorded throughout most of the scheduled wake episodes to document 
wakefulness [107]. The DSR will be connected to the control room via a special extension cord that will enable 
staff in the control room to monitor the signals online.  
 
Sleep restriction. Our laboratory has well-established procedures for ensuring participants in sleep loss protocols 
remain awake throughout the entirety of their scheduled wake episodes [13, 32, 106, 107]. Participants may be 
directly monitored in their study room during the sleep intervention segments to ensure they remain awake and 
adhere to the protocol. The staff members who carry out this monitoring are trained to directly observe the face 
and eyes of the participant and to engage them in conversation or other activity (such as playing a board game) 
if the participant is struggling to remain awake. In the Sleep Restriction without Extended Light condition, the 
participants will be asked to remain awake while sitting in bed in near-darkness for 2.5 hours before and after 
each 5-hour sleep episode during the week of sleep intervention. We have experience ensuring participants 
remain awake even in such challenging conditions. The waking EEG recordings are reviewed on a regular basis 
and feedback is provided to the study staff if brief sleep episodes are detected.    
 
Diet. Appropriate dietary control is important for ensuring that any modulation of glucose metabolism and other 
metabolic measures during the study are due to sleep or light exposure and not dietary changes such as calories, 
sodium, or other parameters that have been shown to modulate glucose metabolism. The diet throughout the 
inpatient portion of the study will be designed to be isocaloric, and the Mifflin St. Jeor equation [96] with a 1.6 
activity factor will be used to calculate the caloric need of each participant. The diet will have a predetermined 
macronutrient percentage (58-60% carbohydrates, 15-17% protein, and 25-27% fat) and a set micronutrient 
intake (calcium 800-1000 mg, potassium 100 mEq [+/-20%], and sodium 150 mEq  [+/-20%]) for all participants. 
The timing and content (macronutrients, %daily calories) of meals will be the same each study day, and between 
participants. Participants will be recruited and screened with full knowledge of the dietary control aspect of the 
study. 
 
Resting Energy Expenditure (REE). REE will be assessed by indirect calorimetry using expired gases toward 
the end of each baseline and toward the end of each sleep intervention segment. Assessments will begin after 
waketime while the participant is fasting. Participants will remain in bed in a semi-recumbent posture while the 
equipment is set up and calibrated. The recording will last for ~15-30 minutes, during which the participant will 
breathe through a disposable mouthpiece into validated and FDA-approved indirect calorimeter (VMAX29 
Encore, Carefusion, or similar device). The calorimeter estimates resting energy expenditure (REE, in kcal/day) 
and RQ (a dimensionless number corresponding to the amount of expired VCO2 per amount of oxygen 

 
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consumed (VO2). Carbohydrate oxidation and lipid oxidation rates can also be calculated (as grams per minute 
[g/min] according to the formulae of Frayn [116], assuming negligible protein oxidation [103-105].  
 
Blood sample collection. The BWH CCI has a system for collecting frequent blood samples from participants 
even while they are sleeping. This system uses an indwelling intravenous catheter placed in a forearm vein by 
a CCI nurse. The catheter is connected to a triple-stopcock manifold via an intravenous loop with a 12-foot small-
lumen extension cable through which blood sampling can continue in the next room without disturbing the 
participant during sleep. Between samples, a solution of 0.45% saline with 5,000 IU/liter of heparin will be infused 
at a rate of 40 cc/hour to maintain patency. This blood sample collection system has been in use by our research 
group for more than 30 years [97]. CCI technical staff and nurses are trained in the collection of multiple, frequent 
blood samples using this system, and it is in routine use on the BWH IPM on a daily basis. We will use this 
system to draw the samples for baseline and end of intervention circadian rhythm assessments, to draw the 
fasted samples and frequent blood samples for the baseline and end of intervention Mixed Meal Responses and 
IVGTT samples, and to draw samples for exploratory -omics analyses.   
 
Standardized Mixed Meal Response test. Post-prandial mixed meal metabolic responses will be assessed 
toward the end of each baseline and toward the end of each sleep intervention segment following the 
measurement of RMR/REE. This will be done after an overnight fast using a breakfast [32]. The meal will have 
the overall composition of the diet described above, but each participant will be served an identical breakfast at 
each test and will be required to eat all of the meal within 30 minutes. Blood samples (approximately 4mL) will 
be collected using the sample collection system described above, with careful attention to the timing of the start 
of the breakfast, and will include pre-meal baseline samples as well as post-prandial samples at ~10-min 
intervals for the first 90 minutes following the meal, then at ~30-min intervals for the next 90 minutes (total of 3 
hours). The participant will remain sitting in bed until the final blood sample is drawn. The samples will be placed 
into collection tubes and centrifuged, then pipetted into small polystyrene tubes and stored at -80°C for later 
analysis of glucose, insulin, and other hormones using standard procedures and/or commercially available kits.  
 
Hormonal data. The recording of the circadian rhythm of melatonin is essential, as it is considered a “gold 
standard” way to assess central circadian phase. The melatonin rhythm has been demonstrated to be the most 
precise and accurate marker of the human circadian pacemaker [98-100]. Samples of blood will be collected at 
approximately hourly intervals through the blood collection system described above. Blood samples 
(approximately 1-3mL) will be transferred to small (3 mL) vacutainer tubes and centrifuged; the resulting plasma 
will be pipetted into vials and frozen until analysis. Melatonin assays will be performed by Solidphase (Portland, 
ME) using a commercially-available radioimmunoassay kit.  
 
Intravenous Glucose Tolerance Test (IVGTT; insulin-modified). An insulin-modified IVGTT will be carried out on 
Days 7 and 15 after an overnight fast [13] and assessment of REE. Blood samples will be drawn via an 
intravenous catheter every 5 min for 20 min starting at T=-20 min.  At time 0 (time of usual breakfast), 0.3 g/kg 
glucose will be administered over 1 minute as an intravenous bolus via an intravenous catheter in the opposite 
(non-sampling) arm.  Blood samples will be taken at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 21, 22, 24, 26, 28, 30, 35, 
40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, and 180 min. At time +20 min, 0.02U/kg human insulin will be 
administered intravenously over 1 minute. Minimal Model Analysis [102] will be used to assess insulin sensitivity 
(SI) and glucose effectiveness. Acute insulin response (AIRg) will be calculated as the average incremental 
plasma insulin concentration over the first 20 minutes after the glucose bolus (and before the insulin 
administration) [22]. 
 
Continuous glucose monitoring. As supplementary/exploratory data, we may use an Abbott FreeStyle continuous 
glucose monitor (Abbott Laboratories) to document glucose levels every 15 minutes throughout the inpatient 
study. If so, a physician or nurse will insert an interstitial glucose sensor in the participant’s arm on Day 4, and it 
will be replaced approximately every 10 days or as needed throughout the end of the study. The sensor will thus 
be worn throughout the study to collect continuous glucose data (one measurement every 15min). The data will 
be downloaded at the end of the collection period using a reader device. This information will provide a dynamic 
view of changes in glucose levels throughout the study, for example in near-darkness while awake.    
 

 
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Biomarkers of sleep loss (panel).  This panel will be collected for exploratory analyses, and reflects known and 
emerging blood and/or saliva constituents that are candidates quantifying the health impact of sleep loss on 
systems including glucose regulation (HbA1c, fasting insulin and glucose [HOMA index], free salivary cortisol 
levels), thyrotropic axis function, lipid metabolism (NEFA release during IVGTT), and peripheral markers of 
energy balance (leptin and ghrelin). Samples may be collected before the start of each IVGTT and assayed for: 
Lipid Panel, HbA1c, insulin-like Growth Factor 1 (IFG-1), thyroid stimulating hormone (TSH), free thyroxine (T4), 
tri-iodothyronine (T3), electrolytes, adiponectin, aldosterone, plasma renin activity (PRA), and/or C-reactive 
protein (CRP).  
 
Plasma lipidomics. Profiling analysis involves giving primary consideration to a collective network of metabolites 
[levels of long-chain highly un-saturated lipids and short-chain, highly saturated lipids], and focusing on the 
determination of categorical differences between physiological states [baseline vs. after sleep restriction, with 
and without ALAN]. Our liquid chromatography-mass spectrometry (LC-MS) method enables separation of lipid 
classes as well as both qualitative and quantitative detection of >600 structurally identified individual lipids in 
biological samples.36-47 Data are acquired using high resolution full scan MS and high energy collisional 
dissociation MS/MS. The method was evaluated for efficient separation and detection in both positive and 
negative ionization mode using standards spanning 6 lipid classes. The approach is currently able to detect fatty 
acids 
(FA), 
sterols 
(ST), 
lysophosphocholines 
(Lyso-PC), 
phosphotydilcholines 
(PC), 
phosphotidylethanolamines 
(PE), 
lysophosphoethanolamines 
(Lyso-PE, 
phosphotydylglycerols 
(PG), 
sphinglipids (SL), diacylglycerols (DG), phosphotidylserines (PS), quinones such as ubiquinone, acyl carnitines, 
cholesterol esters, cholesterol sulfate, sulfatides, hexCers and and other lipid subclasses. We will begin by 
generating lipidomic profiles of all samples, assess levels of individual lipids in baseline vs sleep restricted 
conditions (separately for each light/no light intervention at this phase) looking at both absolute and relative 
signal (ie., mass effect of lipids and relative occupation of total lipid pool). For the triglycerides, we will additionally 
assess the set Rhee et al.1 associated with diabetes risk [e.g., increased risk with TG 44:1; TG 46:1; TG 48:0; 
TG 48:1; TG 50:1; TG 52:1;  decreased risk with TG 56:9, TG 58:10, TG 60:12]; we will then look at total carbons 
and total double bonds in each species in the triglyceride and phospholipid pools vs. the sleep restriction 
phenotypes. We will assess the within-person stability of each lipid within the baseline data itself. Finally, we will 
test the general hypothesis.  
 
Total fat mass and BMI. Body Mass Index (BMI) will be assessed using measured height and weight, and waist 
and hip circumference. A DXA scan (Discovery W Dual-energy X-ray Absorptiometry Scanner, Hologic, Bedford 
MA) will be carried out at the BWH Bone Density and Body Composition Research Core to measure total fat 
mass prior to the start of the study and again at the end of the study. We will look for changes in fat mass, 
especially visceral/trunk adiposity, and will further be able to determine whether there are changes in muscle 
mass with the low levels of activity in controlled laboratory conditions. 
 
Saliva sample collection. Saliva will be collected approximately every waking hour as a backup to the blood 
samples collected for melatonin [100]. Participants are instructed to not eat anything within 30 minutes of a 
sample, and may be asked to rinse their mouth with water after eating. The exact time of collection will be 
documented and the sample will be frozen at -20ºC for later assay (if needed).  
 
Oral microbiome sample collection. Studies have found that oral microbiota are impacted by delayed meal timing, 
which is common in shift workers and people obtaining insufficient sleep. Additional saliva samples and/or tongue 
swabs may be collected approximately once per day throughout the study for salivary/oral microbiome analysis. 
The exact time of collection will be documented and the sample will be frozen at -80ºC for later assay. 
 
Genetic sample collection and whole genome sequencing. Given the findings that genetic polymorphisms are 
associated with sleep, circadian, and metabolic physiology, we will collect genetic material from each participant 
for genome-wide analysis as well as future research. A total of 30mL of blood will be collected from each 
participant either at the time of discharge from the study or during a screening visit. Blood samples will be sent 
to the Mass General Brigham Biobank where DNA will be extracted, purified, and stored [49, 118, 119]. A portion 
of each sample may be sent to the Genomics Platform at the Broad Institute where standard whole genome 
sequencing (WGS) will be performed. 

 
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Objective alertness assessment. At approximately hourly intervals throughout the scheduled wake episodes, 
participants will take a modified Karolinska Drowsiness Test (KDT [107, 120, 121]) to evaluate objective 
alertness. The KDT is a standardized procedure for providing a highly sensitive, objective EOG (incidence of 
slow eye movements and eye blink rates) and EEG measures to assess alertness. During the KDT, participants 
are asked to remain still while fixing their gaze for ~4 minutes, avoiding frequent eye blinks, eye movements and 
body movements. This test allows for reliable collection of EEGs of high signal quality. These objective measures 
of sleepiness have been shown to vary with circadian phase and duration of wakefulness[122-126]. Our staff 
and the technical staff of the CCI routinely conduct such recordings. EEG will also be recorded throughout the 
wake episodes to document wakefulness [107]. 
 
Subjective alertness assessment. Throughout the study, participants will complete computer-administered 
alertness assessments at approximately hourly intervals when they are awake. These will include the Karolinska 
Sleepiness Scale (KSS) and a Visual Analog Scale (VAS). The KSS requires the participant to select a number 
on a scale from 1 to 9 spanning the range from very sleepy to very alert. The task takes less than 1 minute, and 
KSS scores have been shown to oscillate with the core body temperature and plasma melatonin rhythms [109-
111]. Visual Analogue Scales (VAS) consist of a horizontal line drawn on the computer display with each end of 
the line labeled with the extremes of a subjective continuum, e.g. sleepy-alert. Participants indicate a position on 
the line that best describes how they feel at that moment. The task takes less than 1 minute to complete, and 
subjective estimates have been shown to vary with circadian phase and sleep loss [112-115].  
 
Neurobehavioral performance assessment. Throughout the study, participants may be asked to complete 
additional computer-administrated performance test batteries several times per wake period. This battery may 
include the following tests: 
• 
Psychomotor Vigilance Task (PVT) to assess sustained attention performance. The PVT is a test of 
visual reaction time (RT) in which the subject is asked to maintain the fastest possible RTs to a simple 
visual stimulus for ~10 minutes. The inter-stimulus interval varies randomly between 2-10 seconds. 
• 
A Calculation Performance task (a measure of cognitive throughput), modeled after one described 
by Klein et al.. This task presents the subject with a series of randomly generated pairs of 2-digit numbers 
and the subject's task is to sum as many pairs as possible in the allotted four-minute time interval.  
• 
Visual search tasks to investigate attentional selection, including a conjunction search task in which 
the subject must look for a target (red vertical bar) among a variable number of distractors (green vertical 
and red horizontal bars), a serial search task in which the subject must look for a target “5” among a 
variable number of distractors (“2”s), and/or the Comparative Visual Search task (CVS) which includes 
a component of working memory. The task requires subjects to compare two side-by-side images (a 
target and a copy) consisting of an array of triangles pointing left and right. In the “copy” version, the two 
images are exact copies with the exception that one of the triangles in the target diagram is pointing in 
the wrong direction; in the “mirror” version of this task, the two images are mirror images of one another, 
and again the subject’s task is to locate the triangle that is pointing in the wrong direction. Thus, the 
subject must remember which parts of the copy array he is comparing with the target and the direction of 
the triangles within it, and remember which part of the target array he has already searched (working 
memory), as well as searching for the target triangle.  
• 
The Stroop color-naming task, a test of executive function. This test involves the inhibition of a 
prepotent response, and performance on the Stroop has been demonstrated to slow with sleep loss. Our 
version of the Stroop includes three types of stimuli:  matching word and color (congruent), non-word and 
color (neutral), and mis-matched word and color (incongruent)69.  
• 
A Probe Recall Memory (PRM) test, a test of short-term memory for unassociated pairs of words, 
consisting of a 30 second learning phase and then recall (either immediately or after a fixed delay). During 
the learning phase, the subject is required to memorize ~6 pairs of unassociated words. During the delay 
phase, the subject is administered an unrelated cognitive task. In the recall phase, the subject is required 
to recall the second word of each pair in response to the computer’s presentation of the first word or to 
answer yes or no in response to pairs of words presented on the screen. 
• 
A Face-Name Association task (F-N) in which the subject is presented with a series of photographs of 

 
Detailed Protocol, version 11 
 
 
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individuals they do not know during the learning phase, and each photograph has a name associated 
with it. They are tasked with remembering the name of each face. The recall phase of this test occurs 1 
or more hours later. Performance on this task activates the hippocampus and left inferior prefrontal 
cortex., and is impaired in dementia and MCI.  
• 
Matrix Reasoning Test (MRT), in which the subject is shown a series of twelve 3 x 3 matrices in which 8 
of the 9 squares of the matrix are populated with an image and the 9th square (in the lower right of the 
matrix) is left blank. Subjects are also presented with a 2 x 4 matrix that contains the correct image in 
addition to 7 incorrect images and must select the image that completes the 3 x 3 matrix.  
• 
Balloon analogue risk task (BART), which assess risk-taking behavior. Participants are presented with 
30 trials in which they must choose to either inflate an animated balloon presented on the left side of the 
screen or collect a reward. The reward can be collected at the beginning of the trial (without inflating the 
balloon) or after any number of pumps which inflate the balloon by increments. The reward that can be 
collected on each trial is proportional to the final size of the balloon; however, if the balloon is overinflated 
and pops, the reward for that trial is lost.  
• 
Tests of emotional memory and/or perception, such as the Emotional Memory Task in which subjects 
view images, slides, or scenes ranging in emotionality from standardized and validated stimuli sets (e.g. 
International Affective Picture System; IAPS or Nencki Affective Picture System; NAPS) and are asked 
to rate and/or remember the scenes for later testing; the Emotional Trade-off Task, in which subjects 
view images consisting of an object, either emotional (i.e. a car wreck) or neutral (i.e. a taxi), composited 
onto a background (i.e., a city street) and are later shown objects and backgrounds in isolation and asked 
to remember whether or not they saw an identical or similar item; and the Morphed Faces Task, where 
subjects view a series of pictures created by separately morphing a neutral face with 3 different emotional 
faces (Angry, Happy and Sad) and are asked to identify each face’s emotional category, as well as rate 
their interpretation of its intensity and valence (negative to positive).  
Voice recording collection. During scheduled wake episodes a speech task may be administered in addition to 
each scheduled neurobehavioral test battery proposed above. The speech task will be administered via 
computer and will take less than 2 minutes in total. Each speech task begins with a prompt to the participant that 
their voice will be recorded throughout the task, and to make sure they are sitting facing the computer monitor 
and microphone. They are then asked to read a list of 10 sentences from the Harvard Sentences corpus [IEEE 
Recommended Practice for Speech Quality Measurements. IEEE Trans Audio Electroacoust. 1969;17(3):225-
246. doi:10.1109/TAU.1969.1162058].  
 
Steps for a single speech task: 
1. The computer program first generates a random 3-digit number and picks a list (10 sentences) 
from the Harvard Sentences for the current assessment. 
2. One sentence at a time is presented to the subject on the computer monitor. After reading out the 
sentence, the subject presses the spacebar on the keyboard to proceed to the next sentence.  
3. After recording the 10 sentences by repeating Step 2, the 10 recordings are saved separately as 
waveform audio files (WAV) under a folder named by the random 3-digit number from Step 1. 
 
The speech of the subjects will be recorded by a Shure VP64A omnidirectional microphone or equivalent 
(frequency response: 50 to 12,000 Hz) mounted on a desktop microphone stand. The microphone is connected 
to the computer via a USB to XLR adapter and cord extensions. The height and horizontal extension of the stand 
are adjusted to hold the microphone close to the lips of the subject when sitting at the desk in their study room  
in the lab. The microphone is covered by a standard windscreen. 
 
While previous studies used different text corpora and languages when recording human voice, most have 
emphasized phonemically balanced texts [Bagnall AD, Dorrian J, Fletcher A. Some Vocal Consequences of 
Sleep Deprivation and the Possibility of “Fatigue Proofing” the Voice With Voicecraft® Voice Training. Journal of 
Voice. 2011;25(4):447-461. doi:10.1016/j.jvoice.2010.10.020; Icht M, Zukerman G, Hershkovich S, et al. The 
“Morning Voice”: The Effect of 24 Hours of Sleep Deprivation on Vocal Parameters of Young Adults. Journal of 
Voice. 2020;34(3):489.e1-489.e9. doi:10.1016/j.jvoice.2018.11.010; Amato F, Rechichi I, Borzi L, Olmo G. Sleep 
Quality through Vocal Analysis: a Telemedicine Application. In: 2022 IEEE International Conference on 

 
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Pervasive Computing and Communications Workshops and Other Affiliated Events (PerCom Workshops). IEEE; 
2022:706-711. doi:10.1109/PerComWorkshops53856.2022.9767372] and vowel pronunciations [Barron DS, 
Heisig S, Agurto C, et al. Feasibility Analysis of Phenotype Quantification from Unstructured Clinical Interactions. 
Computational Psychiatry. 2022;6(1):1. doi:10.5334/cpsy.78; Rahman W, Lee S, Islam MS, et al. Detecting 
Parkinson Disease Using a Web-Based Speech Task: Observational Study. J Med Internet Res. 
2021;23(10):e26305. doi:10.2196/26305]. Thus, our speaking task involves reading one list from the Harvard 
sentences [IEEE Recommended Practice for Speech Quality Measurements. IEEE Trans Audio Electroacoust. 
1969;17(3):225-246. doi:10.1109/TAU.1969.1162058] each time. There are 72 lists of Harvard sentences, each 
containing 10 sentences. The computer program will pick a distinct list from the corpus every time the speech 
task is performed so subjects do not become familiar with the text and degrade their pronunciation by reading 
too fast or rumbling. 
 
Stool sample collection. The gut microbiome is thought to be a potential candidate mechanism underlying the 
effects of sleep and circadian disruption on metabolic health. Therefore, we may collect stool samples at several 
points throughout the study for use in microbiome analysis. On specific study days, participants may be instructed 
to notify the staff at their next bowel movement so that they may be provided with a collection kit for stool 
sampling. The exact time of sampling will be documented and the sample will be frozen at -20ºC for later assay.  
 
Breath sample analysis. We may use selected ion flow tube mass spectrometry (SIFT-MS) to collect samples of 
breath for metabolomic analysis. The Syft T007 Voice200 Ultra performs Selected Ion Flow Tube Mass 
Spectrometry (SIFT-MS) and is a technology that has the ability to identify breath biomarkers accurately and 
reliably. Syft is sensitive (up to parts per billion), specific (~1000 known compounds), fast (sampling time <1 
minute), and inexpensive (requiring only plastic straws and water to acquire a sample). At selected intervals 
during the study, the participant may be asked to take part in a breath collection segment. They may be asked 
to brush their teeth with water and/or rinse their mouth, and then be seated and remain seated for the 20-30 
minute collection. During the sample collection segment, the participant will be asked to inhale through their nose 
and exhale into a straw connected to the Syft T007 Voice200 Ultra for ~2.5-minute sampling segments.  
 
Private Identifiable Information. All data for this study will be collected and recorded using a study code, rather 
than individually-identifying information. This is a routine practice in our laboratory and at the CCI, and we 
therefore have systems in place to assign a study code upon enrollment into the protocol and to use study codes 
on automated data collection systems in the CCI (for the activity data, PSG data, alertness and performance 
data, biospecimen, breath samples).  
 
Some identifiable information about the study will be collected. Tests and questionnaires from the screening 
process do include identifiable information. The Division of Sleep and Circadian Disorders has procedures in 
place to maintain the confidentiality of that information in areas with limited access. During the inpatient study at 
the CCI the participant is considered a patient at the hospital, so record of their participation and limited records 
of their visit (admission and discharge notes, CCI RN and attending MD notes, daily vital signs, clinical blood 
test results) are including in their electronic medical record. No study data are kept in the medical record. As 
outlined in the Human Subjects section below, all staff members of the Division of Sleep and Circadian Disorders 
and the CCI are trained in HIPAA compliance, research ethics, and privacy issues, and are routinely re-certified.  
 
VI. 
Biostatistical analysis 
 
Intent-to-treat analysis and per protocol analysis will both be performed. The amount and patterns of missing 
data, if any, will be characterized. We will use all available data points in the mixed-effects model. The study will 
be monitored routinely for issues of data quality, study conduct, and adverse events. We do not plan to do interim 
analysis. Mediation analysis will be performed to study the relationship between extended-duration ALAN 
exposure, duration of melatonin secretion, and the primary outcomes (insulin sensitivity and glucose tolerance). 
Paired t-test (or Wilcoxon signed-rank test) will be used to compare the change from baseline in insulin sensitivity 
(SI), acute insulin response (AIRg), glucose effectiveness, glucose AUC, insulin AUC, and the duration of the 
melatonin profile after exposure to sleep restriction with and without ALAN. Linear mixed models will also be 
used to compare the change in these outcomes from baseline to after exposure to sleep restriction with and 

 
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without ALAN in case of missing data and with potential covariates, such as age and sex.  
 
Insulin sensitivity (SI) (Specific Aim 1). An insulin-modified IVGTT will be carried out on Days 4 and 11 after an 
overnight fast [13]. Blood samples will include pre-test baseline samples. At time 0 (time of usual breakfast), 0.3 
g/kg glucose will be administered over 1 minute.  Blood samples will be taken every minute for the initial 6 
minutes, every ~2 minutes for the following ~20 minutes. At time +20 min, 0.02U/kg human insulin will be 
administered intravenously over 1 minute. Blood samples will continue to be taken every 5 minutes for the 
following half hour, every 10 minutes for the next ~half hour, and every 20 minutes for the next 1.5 hours. Minimal 
Model Analysis 3 will be used to assess insulin sensitivity (SI). 
 
Acute insulin response (AIRg) (Specific Aim 1). An insulin-modified IVGTT will be carried out on Days 4 and 11 
after an overnight fast [13]. Blood samples will include pre-test baseline samples. At time 0 (time of usual 
breakfast), 0.3 g/kg glucose will be administered over 1 minute.  Blood samples will be taken every minute for 
the initial 6 minutes, every ~2 minutes for the following ~20 minutes. At time +20 min, 0.02U/kg human insulin 
will be administered intravenously over 1 minute. Acute insulin response (AIRg) will be calculated as the average 
incremental plasma insulin concentration over the first 20 minutes after the glucose bolus (and before the insulin 
administration) [22]. 
 
Glucose effectiveness (Specific Aim 1). An insulin-modified IVGTT will be carried out on Days 4 and 11 after an 
overnight fast [13]. Blood samples will include pre-test baseline samples. At time 0 (time of usual breakfast), 0.3 
g/kg glucose will be administered over 1 minute.  Blood samples will be taken every minute for the initial 6 
minutes, every ~2 minutes for the following ~20 minutes. At time +20 min, 0.02U/kg human insulin will be 
administered intravenously over 1 minute. Additional blood samples will be drawn at minutes 21, 22, 24, 26, 28, 
30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, and 180.  Minimal Model Analysis [102] will be used 
to assess glucose effectiveness.  
 
Glucose AUC in response to a standardized Mixed Meal (Specific Aim 2). Post-prandial glucose response will 
be assessed at breakfast following an overnight fast [32] at baseline (Day 3) and at the end of the sleep 
intervention segment (Day 10). The meal will be identical at each test and the participant will be required to eat 
all of the meal within 30 minutes. Blood samples will include pre-meal baseline samples as well as post-prandial 
samples at 10-min intervals for the first 90 minutes following the meal, then at 30-min intervals for the next 90 
minutes. The glucose area under the curve for time 0 to 120 minutes will be calculated using the trapezoidal 
method. 
 
Insulin AUC in response to a standardized Mixed Meal (Specific Aim 2). Post-prandial insulin response will be 
assessed at breakfast following an overnight fast [32] at baseline (Day 3) and at the end of the sleep intervention 
segment (Day 10). The meal will be identical at each test and the participant will be required to eat all of the meal 
within 30 minutes. Blood samples will include pre-meal baseline samples as well as post-prandial samples at 
10-min intervals for the first 90 minutes following the meal, then at 30-min intervals for the next 90 minutes. The 
early phase insulin area under the curve for time 0 to 30 minutes will be calculated using the trapezoidal method. 
The late phase insulin area under the curve for time 30-120 minutes will also be calculated using the trapezoidal 
method. 
 
Analysis of circadian phase (Specific Aim 3). Plasma melatonin will be collected throughout the circadian 
phase assessment procedures on Days 3-4 and 10-11 for assessment of circadian phase and amplitude. Two 
measures of melatonin phase can be derived from the data collected in the proposed studies. In the first 
method, the dim light melatonin onset (DLMO), the time at which the plasma levels reach 10 pg/ml, will be 
determined [130]. To account for the wide variation between individuals, a second method of estimating 
melatonin phase, a relative dim light melatonin onset (DLMO25%) will also be estimated, computed as the time 
at which the plasma melatonin rhythm crosses a threshold of 25% of its fitted value [98, 100] derived from the 
entire baseline time series. Linear interpolation between adjacent samples will be used to determine DLMO 
and DLMO25%. The same threshold derived from the melatonin data on the baseline profile will be used to re-
assess DLMO25% on the re-assessment (at the end of the sleep intervention). In addition, the area under the 

 
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study will be exposed to room light for an extra 5 hrs/day in the sleep restriction with ALAN condition, we expect 
to see a more than 3-hour difference in the duration of melatonin secretion with and without ALAN.   
 
For Exploratory Aim 4, we will consider two primary outcomes. We estimate that we have 85% power (α=0.025, 
2-sided paired-sample t-test) to detect an 20% difference in the change (25% with ALAN, SD=28% vs. 5% 
without ALAN,SD=6%) in levels of long-chain, highly unsaturated triglycerides, which have been associated with 
insulin sensitivity. Moreover, we estimate we will have 85% power to detect a difference as little as 11% in the 
change (5% with ALAN, SD=6% vs. 18% without ALAN,SD=19%) in levels of short-chain, highly saturated 
triglycerides, levels of which have been associated with increased diabetes risk.  
 
VII. 
Risks and Discomforts 
Potential Risks to Subjects Associated with Each Study Intervention, Procedure or Interaction.  
 
Risks of blood sampling procedures.  
Common Risks 
   There may be some discomfort or bruising on initial insertion of the IV catheter into a vein but wearing the 
catheter should not be painful.  
   Occasionally, there is a black and blue mark at the site of the IV insertion, which may last a couple of weeks; 
and, rarely, a small scar may remain permanently at the venipuncture site.  
   Occasionally, mild discomfort may occur from the tube in the vein. If this happens, we will either reposition it 
or remove it entirely, asking the participant's permission before any subsequent reinsertion.  
   There may be a minor skin rash or reaction (contact dermatitis) to the sterile tape used to hold the catheter in 
position. Hypoallergenic tape will be used as necessary.  
   The amount of blood drawn should not significantly alter blood volume, although there may be a small decrease 
in the hemoglobin level. The total volume of blood drawn over the course of the month-long study will be no more 
than 2 pints (946 mL).  
 
Uncommon Risks 
   There is the possibility that the participant may faint during or after the IV insertion procedure.  
   There is a rare possibility of developing a small blood clot, inflammation, or local infection around the vein 
where the catheter is inserted. In rare cases, a generalized infection can spread through the bloodstream as a 
result of the IV catheter.  
   There may be some side effects from the use of heparin, such as bleeding or allergy. The amount of heparin 
used is much less than what would be used therapeutically to prevent clotting.  
 
Risks of PSG collection.  
Common Risks 
There may be a minor skin rash or reaction (contact dermatitis) at the sites where the electrodes are placed in 
response to the conducting gel, the collodion used to adhere the electrodes to the hair, or to the sterile tape used 
to hold the electrodes to skin on the face.  
 
Risks of insulin-modified IVGTT.  
Common Risks 
   There is a second IV inserted for the IVGTT, and the same risks apply to it as to the sampling IV described 
above.  
   During the IVGTT, the sampling IV line is placed in a hand/wrist vein and this hand may be placed in a heated 
box to allow for easier sampling (140 degrees Fahrenheit). This may cause sweating and slight swelling of the 
hand, and before the IVGTT procedure begins the subject will need to take off any rings that he/she normally 
wears on fingers of the hand being used. The temperature of the heated box and the subject’s hand will be 
closely monitored throughout the entire IVGTT procedure. The subject is free to remove the hand from this box 
if there is discomfort during the procedure. 
 
Uncommon Risks 

 
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   Intravenous glucose infusion will raise blood glucose levels mildly. There is a rare risk of tissue extravasation 
if glucose is pushed too quickly through a peripheral IV line. Accordingly, the current IVGTT protocol used on 
the CCI requires a slow IV push method by an experienced provider who will monitor for pain/tenderness at the 
infusion site. A specially trained clinician will perform the IVGTT, which have been performed over 100 times in 
healthy individuals by our research core. 
   Insulin is injected intravenously in a quantity related to body weight. Insulin lowers blood glucose and can 
cause a lowering of circulating potassium levels. In lowering glucose levels, there is risk of hypoglycemia. 
Symptoms of hypoglycemia are reversed rapidly with correction by either oral or IV glucose administration. The 
IVGTT will be carried out by a specially-trained clinician, and a second nurse will be present throughout the 
procedure. A symptom check is performed at 30, 60, 120, and 180 minutes after glucose infusion. Blood glucose 
safety checks are performed 20 minutes before the start of the procedure, as well as 160 min and 180 min after 
glucose infusion. An “Emergency Medication Kit” is ordered at bedside for each IVGTT that consists of: glucose 
tablets, IV glucose, IM glucagon, IM epinephrine, IV diphenhydramine, IV hydrocortisone, and acetaminophem. 
A CCI Standard Operating Procedure dictates threshold values for glucose and corresponding treatment options.  
   The dose of insulin used in this procedure is small and would not be expected to lead to low potassium levels. 
As a precaution, the participant’s potassium level will be checked to ensure it is within normal range. 
 
Radiation risks 
   Body composition measurement involves radiation exposure from a scan of the whole body using dual x-ray 
absorptiometry or DXA. The radiation exposure from these scans is less than 1% of the normal, annual 
background radiation exposure. The risk from radiation exposure of this magnitude is too small to be detected 
in this study's population. 
 
Risks of receiving study visit reminders by text message 
 Text messages are not encrypted, and thus there is a risk of a loss of privacy. To minimize this risk, we will 
adhere to the principles of minimum necessary; thus, study-initiated text messages will not contain participant 
names or other identifiers, sensitive information that could expose financial risk to individuals (social security 
numbers, driver license numbers, credit card numbers, etc), results of any clinical tests conducted for the purpose 
of research, or potentially sensitive information about the study visit. Additionally, participants who consent to 
receiving text messages will first be sent a welcome message by text that requests the participant to respond 
“yes” to indicate their preference to continue to receive research texts.  
 
Other Study Risks.  
Common Risks 
   Staff in the CCI metabolic kitchen will calculate a menu cycle based on the individual participant's preferences 
that meets the diet order and is designed to maintain each participant’s weight and adhere to the planned 
macronutrient content. The participant will be required to eat all the foods they are served at each meal. 
Furthermore, on each of the occasions that a Mixed Meal Response test is given, the content of that breakfast, 
and the food on all the meals on the prior day, will be exactly the same. The participant may find this to be 
unappealing, but they will be consuming foods from a menu of study-compatible options that they choose. 
   For all scheduled sleep episodes, and for the 2.5 hours before and after each restricted sleep episode in the 
Sleep Restriction without ALAN condition, the participant will be restricted to bed and will not be allowed to get 
out of bed. During scheduled sleep episodes, the room will be completely dark. The participant will not be allowed 
to leave the bed or turn on the lights during the scheduled sleep episodes, even if they find it difficult to remain 
asleep. They will have to use a bedpan or urinal as needed during this time. 
   For the assessment of Resting Energy Expenditure and the Mixed Meal Response baseline tests, the 
participant will be restricted to sitting quietly in bed from the time that they are awakened until the blood sampling 
from the Mixed Meal test is complete approximately 4 hours later. For the same tests carried out at the end of 
each sleep restriction intervention condition, participants will awaken 2.5 hours before usual, and thus will remain 
sitting in bed for approximately 6.5 hours. The participant will have to use a bedpan or urinal as needed during 
this time. 
   For the 2.5 hours before and after each restricted sleep episode in the Sleep Restriction without Extended 
Duration ALAN condition, the lighting in the room will be very dim. This will make it difficult for the participant to 

 
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read or do other visual tasks. There will be a trained staff member in the room with them to converse, play a 
board game, etc. 
   There will be times when the participants will feel extremely sleepy. There are no known permanent adverse 
effects from chronic sleep restriction for a limited time such as the 2 weeks in the proposed studies. The 
participant may experience headache, nausea, upset stomach, or feel very irritable or frustrated when they are 
very sleepy and not allowed to go to sleep. The participant will be informed that they may withdraw from the 
study at any point that they feel this sleepiness is too extreme, but they will be required to sleep for at least 4 
hours before being discharged from the study. 
   The assessment of resting metabolic rate will take approximately 20-30 minutes, during which time the 
participant will be asked to sit underneath a ventilated hood. This may be unpleasant for some individuals. If the 
subject/patient feels that the discomfort is excessive he/she can choose to terminate the study. 
Body composition measurement will be done by a scan of the whole body using dual x-ray absorptiometry or 
DXA. This may involve the minor discomfort of lying still for 5 minutes.  
Mild discomfort may occur when the continuous glucose monitor sensor is first placed. It should not be 
uncomfortable while wearing it. Mild skin irritation may occur when the sensor is removed at the end of the 
sampling segment. 
Mild irritations such as edema, rash, bleeding, itching and infection may occur from the site where the 
continuous glucose monitor sensor is placed. In our study, the sensor will be placed by a nurse after cleaning 
the skin in order to minimize the risk of infection. The site will be checked daily by a CCI nurse and the placement 
moved if edema, rash, itching, or bleeding occurs. 
   Participants may feel uncomfortable answering some of the questions that are part of the pre-study screening 
process. 
   Participants may feel uncomfortable answering some of the questions that are part of the hospital admission 
process. 
   Participants may feel uncomfortable viewing the emotional imagery used in some of the performance tests.  
   Participants may feel uncomfortable having a staff member in their room throughout the entire wake episodes 
during the week of sleep intervention. They will be informed of this monitoring ahead of time, and can choose to 
end the study early if they feel they cannot tolerate it. 
   Because of the sleep loss experienced during the study, participants will be advised that they will likely be 
sleepier than usual after the study is complete, and that this may last for several days. They will be advised to 
allow themselves extra time for sleep and to avoid situations that put them at risk for an accident, such as driving 
an automobile or operating heavy machinery.  
   In order to minimize the risks associated with jet-lag type symptoms, participants will be counseled prior to 
discharge on how to readjust to their regular sleep-wake cycle if they should experience jet lag symptoms. In 
our experience, most potential participants have either experienced jet-lag in the past, and/or have kept an 
irregular schedule in the past, and thus can anticipate what these symptoms will be like. 
 
VIII. Potential Benefits. Although there will be no direct physical benefit to the participant resulting from 
participation in this study, we will make known to the participant, upon conclusion of the protocol and analysis of 
the data, some of the information we have gathered from the physiological testing. This information may help 
the participant develop a more compatible daily schedule for sleeping, which in turn may result in improved well-
being. We will also provide the participant with copies of the screening blood and urine tests, ECG, and physical 
examination form if they request them. 
 
There is a chance that the pre-study screening will reveal some medical abnormality. This information will be 
conveyed to the participant at once, together with a recommendation of a local clinic or physician from whom to 
seek treatment.  
 
The research has the potential to benefit society more widely. This study is designed to evaluate the extent to 
which circadian disruption induced by longer exposure to artificial light at night contributes to the impairment of 
glucose metabolism associated with sleep restriction. By the end of the 20th century, Americans were sleeping 
on average 2 hours less per night than they were at the beginning of the 20th century. In fact, recent polls 
conducted by the National Sleep Foundation indicate that young and older adults alike report sleeping less than 
what is recommended [127, 128]. More than a third of adults report sleeping less than 7 hours per night, with 

 
Detailed Protocol, version 11 
 
 
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 May 23, 2023 
approximately 15% of adults reporting less than 6 hours of sleep per night on a regular basis. While sleep 
researchers have begun to understand the consequences of acute sleep loss on alertness and performance, far 
less is understood about the consequences of chronic insufficient sleep [129] or how to mitigate those impacts. 
While the adverse metabolic consequences of chronic sleep insufficiency are now widely appreciated, the role 
played by light exposure at night on those metabolic changes is not understood. By understanding the 
contribution of extended light exposure on the adverse metabolic changes associated with chronic sleep 
restriction, we can design and test strategies to minimize or eliminate those risks.   
 
Experimental studies have demonstrated that chronic sleep restriction impairs glucose metabolism, consistent 
with epidemiologic studies that show strong links between short sleep, metabolic syndrome, and diabetes. 
However, less is known about whether one of the mechanism(s) by which insufficient sleep impairs glucose 
metabolism is light exposure at night. Given that sleep restriction is typically accompanied by longer exposure 
to artificial light at night, which is known to disrupt circadian rhythmicity, we plan to evaluate the extent to which 
circadian disruption induced by exposure to extended duration artificial light at night contributes to the impairment 
of glucose metabolism associated with sleep restriction. This knowledge can be used to design and test 
countermeasures and mitigation strategies to minimize the adverse metabolic consequences of chronic 
sleep restriction. 
 
IX. Monitoring and quality assurance 
Overall Framework for Safety Monitoring. This study will be conducted under the regulations and oversight of 
the Mass General Brigham (MGB, formerly Partners HealthCare System) Human Subjects Committee, required 
for all studies carried out at the Brigham and Women’s Hospital. The PI (Dr. Czeisler) will be responsible for 
ensuring all Human Subjects regulations and policies are followed, and for reporting to the Human Subjects 
Committee, as required. Furthermore, the study must be approved by the Harvard Catalyst Clinical and 
Translational Science Center Scientific Advisory Committee before it can be conducted on the BWH CCI. All 
study personnel involved in the participant recruitment, study execution, data collection, and data analysis at 
BWH have completed Human Subjects training as well as training in the procedures required by HIPAA, and 
must annually certify that they will comply with all such regulations. Drs. Czeisler, Duffy, Kristal, Saxena, Scheer, 
Wang, Williams, and Yuan have completed additional training in Medical Ethics as required by their appointments 
at the Harvard Medical School.  
 
Individual(s) or Group Responsible for Monitoring. The PI will be responsible for carrying out the DSMP. 
Data and safety will be monitored by the PI, with assistance from the study personnel, attending physicians, and 
CCI nurses, and reported to the IRB as required. The procedures proposed in this study have been in use in the 
PI’s research program for the past 40 years, and his team as well as the CCI research nurses and technicians 
have used the same procedures in hundreds of studies over the two decades since the CCI-IPM opened.  
 
Information to be Monitored. The following aspects of the study will be monitored: enrolment, including accrual 
and retention and tracking by sex and race/ethnicity; protocol non-compliance; data completeness; and adverse 
events.  
 
Specific issues to be monitored include: adherence to the sleep schedule, meal diary, and wearing of the 
actigraphy monitor in the pre-study phase; collection of the blood samples and compliance with the conditions 
for circadian analysis; conduct of the Mixed Meal response procedure including collection of the frequent blood 
samples; conduct of the intravenous glucose tolerance tests including collection of the frequent blood samples, 
and occurrences of hypoglycemia; collection of the PSG data each night of the inpatient study; collection of EEG 
and EOG data during the wake episodes to verify wakefulness throughout all scheduled times; collection of 
vigilance and subjective sleepiness data during the scheduled wake times; collection of voice recordings during 
scheduled wake times, including suitable volume and clarity for proposed audio analyses, enough sentence 
completeness, and the correct file format (WAV) and storage location; common and uncommon risks as 
described elsewhere. 
 
Procedures for Monitoring and Frequency of Review. During the inpatient study conducted on the CCI, 
participants will be supervised by CCI nursing staff and technical laboratory personnel to monitor participant 

 
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safety. The CCI nursing staff will make daily evaluations of the participant’s general health and tolerance of the 
study protocol, and will insert and maintain the blood sampling IV lines following CCI SOPs. A nurse or physician 
will insert the continuous glucose monitor probe. A specially-trained CCI nurse and a nurse practitioner will be 
present throughout the intravenous glucose tolerance tests. A physician will perform an admission physical 
examination, will review medical and safety data on a daily basis during the inpatient study, and will be available 
on-call to handle any medical emergency that might arise.  
 
A study team member (postdoctoral fellow) will meet with the participant each day to review their study 
experience; she will meet with the CCI staff to review their impressions of study progress; and she will review all 
data collection for timeliness, completeness, and accurate documentation. Moreover, Dr. Duffy and the 
postdoctoral fellow on the study team will also be on-call throughout the study to answer questions from CCI 
staff. Much of the data (PSG, waking EEG, blood sample times, saliva sample times, lights out and lights on 
times, subjective alertness assessments, voice data) are collected by an integrated data collection system that 
time stamps each piece of information, puts it into a secure database according to participant code, and is backed 
up automatically at least daily. Other study data (diet content, assay results, demographic information, REE 
results) are entered into a secure database using Standard Operating Procedures and double-checked for quality 
control. 
 
Ongoing study progress will be reviewed weekly by the study team. During these meetings, the Principal 
Investigator and other members of the research team are present. At these meetings, protocol deviations and 
incomplete data are reviewed, and decisions are made regarding inclusion/exclusion and individual participant 
termination; such decisions (individual participant termination) that need to be considered in between the study 
team weekly meetings are made via conference call.  
 
If required by the NIH, the PI will submit an annual progress report that confirms adherence to the DSMP, 
including a summary of any data and safety monitoring issues that occurred since the previous reporting period; 
and describing any changes in the research protocol or the DSMP that may or does affect risk. He will also 
provide the NIH with all new and continuing IRB approvals during the course of the project if requested. 
 
How Participant Confidentiality will be Protected. We and the BWH CCI have multiple steps to protect 
participant confidentiality. The MGB Human Research Committee requires investigators of NIH-funded human 
research studies to use a special Certificate of Confidentiality Research Consent Form. 
 
Safety Monitoring. A Safety Officer will be appointed for the study. We will select a licensed physician from 
outside the Division of Sleep and Circadian Disorders as the Safety Officer to avoid any potential conflict of 
interest. The Safety Officer’s CV will be provided to the NIDDK Program Officer prior to appointment for their 
review.   
 
Process by which Adverse Events (AEs), including Serious Adverse Events (SAEs and Unanticipated 
Problems (UPs), will be Managed and Reported. Dr. Williams will make the final determination related to any 
Adverse Events for reporting purposes, including the severity of any event, nature of the event, and relationship 
of the event to study procedures, taking information from the CCI nursing staff and any other medical personnel 
present during an event (such as the specially trained nurses present during the IVGTT procedures) and 
available data and documentation related to the event. Using this information, the PI will then follow IRB, NIH, 
and NIDDK guidelines for reporting the adverse event.  
 
Timeline for Reporting Serious Adverse Events (SAEs and Unanticipated Problems (UPs). Any adverse 
event will be reported to the IRB as required within the time frame required to do so (typically 5 working days; 
serious or unexpected adverse events (AEs) will be reported immediately). The IRB in turn will work with the PI 
to report the serious adverse events (SAEs) to the NIH Program Officer. Any action by the IRB or the CCI that 
results in temporary or permanent suspension of the study will be reported immediately to the NIH Program 
Officer. Minor deviations and non-serious AEs are reported to the IRB annually as part of the protocol continuing 
review process.  
 

 
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Screening Laboratoy Tests and Acceptable Ranges
Description
Normal Values
Acceptable Values
Comments
Comprehenstive Metabolic Panel
SST, 8.5mL
Glucode (Random) Serum Glucose 
54-118mg/dl
54-150mg/dl
Non-fasting. If >150 mg/dl then repeat fasting
Glucose (Fasting)
Serum Glucose 
54-118mg/dl
54-125 mg/dl
Urea
BUN
9-25 mg/dl
6-50 mg/dl
Creatinine
Serum Creatinine
0.7-1.3 mg/dl
0.4-1.4mg/dl
Sodium
Serum Sodium
136-142 mmol/L
3.5-5.0 mmol/L
Potassium
Serum Potassium
3.5-5.0 mmol/L
3.5-5.0 mmol/L
Chloride
Serum Chloride
98-108 mmol/L
94-114 mmol/L
Total CO2
Serum Total CO2
23-32 mmol/L
22-36 mmol/L
Anion Gap
3-15 mmol/L 
2-16 mmol/L
ALT/GPT
Liver Enzyme
7-52 U/L
5-60 U/L
AST/GOT
Liver Enzyme
9-30 U/L
5-40 U/L
Alk Phos
Liver Enzyme
36-118 U/L
20-135 U/L
Total Bili
Liver Function
0.2-1.2 mg/dl
0.0-1.9 mg/dl
1.2-1.9 mg/dl acceptable as long as other LFTs are WNL
Total Protein
Serum Protein
6.0-8.0 g/dl
5-8.5 g/dl
Albumnin 
Serum Protein
3.7-5.4 g/dl
3.5-5.9 g/dl
Globulin
Serum Protein
2.0-4.0 g/dl
1.6-4.1 g/dl
Calcium
Serum Calcium
8.8-10.5 mg/dl
8.7-10.5 mg/dl
Complete Blood Count with Differential
EDTA, 3mL
WBC
White Blood Cells
4-10 Kl/uL
3-10.5 K/uL
RBC
Red Blood Cells
3.9-6.0M.ul
3.0-7.0M/ul
HCT (women)
Hematocrit
36-48 g/dl
34-48 g/dl
HCT (men)
Hematocrit
38-49 g/dl
36.5-49 g/dl
HGB (women)
Hemoglobin
11.5-16.4 g/dl
11.0-16.4 g/dl
HGB (men)
Hemoglobin
12.5-16.6 g/dl
12.0-16.6 g/dl
MCV
Blood MC volume
80-95 um3
76-100 um3
MCH
Blood MCH
27-32 uug
24-36 uug
MCHC
Blood Index
32-36 g/dl
30-38 g/dl
RDW
Blood Index
10-14.5
9-15.5
PLT
Platelets
150-450 K/ul
140-500 K/ul
Lymph %
Lymphocyte %
18-41
10-60%
Mono %
Monocyte %
2.5-8.5
1.8-14
Neut %
Neutrocyte %
48-76
32-84
EOS %
Eosinophil %
0-5
0-9
Baso %
Basophil %
0-2.5
0-9
Lymph #
Lymphocyte Absolute
0.8-4.1 K/ul
0.4-5.8 K/ul
Mono #
Monocyte Absolute
0.10-0.80 /ul
0.02-1.4 K/ul
Neut #
Neutrocyte Absolute
3.9-7.6 K/ul
2.9-9.0 K/ul
Eos #
Eosinophil Absolute
0.0-0.5 K/ul
0.0-0.6 K/ul
Baso #
Basophil Absolute
0.0-0.15 K/ul
0.0-0.3 K/ul
TSH
Thyroid Stim Hormone
0.5-5.0
0.5-5.0
SST, 3.5mL
Urinalysis
COLOR
clear/yellow
clear/yellow/amber/cloudy
SP GR
Specific Gravity
1.003-1.035
1.003-1.045
PH
pH
4.5-8.0
4.3-8.5
PRO
Protein
Negative 
1+
KET
Ketone
Negative 
1+
BILI
Bilirubin
Negative
trace
BLOOD
Blood
Neative
negative (Males) 1+ (Females)
LEUK
Leukocyte
Negative
No more than one
ES
Esterase
Negative
of these may be
NIT
Nitrates
Negative
positive
URO
urobilinogen
0.2-1.0
0.2-1.2
WBC
white blood cells
0-4/hpf
<10
RBC
red blood cells
0-2/hpf
0-2 (males) <10 (females)
BACT
bacteria
negative
1+ (unless sq epitherial cells present)
SQ EPI
squamous epithelial cells variable
variable
Drug Screen
Comprehensive Drug Analysis (Urine)
Negative
Negative
If positive for THC, test may be repeated once
Prengancy Test (Urine)
Negative
Negative
Nicotine Metabolite
Negative
Negative
Alcohol/ETG
Negative
Negative
Caffeine
Negative
Negative
Test results that fall outside the "Acceptable" range can be repeated once to confirm findings