Repeating patterns of sleep restriction and recovery: Do ...€¦ · Full-length Article Repeating patterns of sleep restriction and recovery: Do we get used to it? Norah S. Simpsona,

Brain, Behavior, and Immunity xxx (2016) xxx–xxx

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Brain, Behavior, and Immunity

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Full-length Article

Repeating patterns of sleep restriction and recovery: Do we get usedto it?

http://dx.doi.org/10.1016/j.bbi.2016.06.0010889-1591/� 2016 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: [email protected] (M. Haack).

Please cite this article in press as: Simpson, N.S., et al. Repeating patterns of sleep restriction and recovery: Do we get used to it?. Brain Behav. I(2016), http://dx.doi.org/10.1016/j.bbi.2016.06.001

Norah S. Simpson a, Moussa Diolombi b, Jennifer Scott-Sutherland b, Huan Yang b, Vrushank Bhatt b,Shiva Gautamb, Janet Mullington b, Monika Haack b,⇑aDepartment of Psychiatry and Behavioral Sciences, 401 Quarry Rd., Stanford University School of Medicine, Stanford, CA, United StatesbDepartment of Neurology, Beth Israel Deaconess Medical Center/Harvard Medical School, DANA-727, 330 Brookline Ave., Boston, MA 02215, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 April 2016Received in revised form 18 May 2016Accepted 2 June 2016Available online xxxx

Keywords:Sleep restrictionStressInflammationCortisolSubjective

Despite its prevalence in modern society, little is known about the long-term impact of restricting sleepduring the week and ‘catching up’ on weekends. This common sleep pattern was experimentally modeledwith three weeks of 5 nights of sleep restricted to 4 h followed by two nights of 8-h recovery sleep. In anintra-individual design, 14 healthy adults completed both the sleep restriction and an 8-h control condi-tion, and the subjective impact and the effects on physiological markers of stress (cortisol, the inflamma-tory marker IL-6, glucocorticoid receptor sensitivity) were assessed. Sleep restriction was not perceivedto be subjectively stressful and some degree of resilience or resistance to the effects of sleep restrictionwas observed in subjective domains. In contrast, physiological stress response systems remain activatedwith repeated exposures to sleep restriction and limited recovery opportunity. Morning IL-6 expressionin monocytes was significantly increased during week 2 and 3 of sleep restriction, and remainedincreased after recovery sleep in week 2 (p < 0.05) and week 3 (p < 0.09). Serum cortisol showed a signif-icantly dysregulated 24 h-rhythm during weeks 1, 2, and 3 of sleep restriction, with elevated morningcortisol, and decreased cortisol in the second half of the night. Glucocorticoid sensitivity of monocyteswas increased, rather than decreased, during the sleep restriction and sleep recovery portion of eachweek. These results suggest a disrupted interplay between the hypothalamic-pituitary-adrenal andinflammatory systems in the context of repeated exposure to sleep restriction and recovery. The observeddissociation between subjective and physiological responses may help explain why many individualscontinue with the behavior pattern of restricting and recovering sleep over long time periods, despitea cumulative deleterious physiological effect.

� 2016 Elsevier Inc. All rights reserved.

1. Introduction

Patterns of restricting sleep during the week and ‘catching up’over the weekend are prevalent in modern society (Hansen et al.,2005; Monk et al., 2000; National Sleep Foundation, 2010; Tsuiand Wing, 2009; Wing et al., 2009). These sleep patterns are notcommonly thought of as deleterious; however, there is limitedempirical evidence to support this belief. Given the wealth of accu-mulated evidence that insufficient sleep is associated with ele-vated health risks (e.g., cardiovascular disorders (Grandner et al.,2013), metabolic disorders (Knutson et al., 2007), and chronic painconditions (Finan et al., 2013)), gaining a better understanding ofthe impact of these common sleep patterns is essential.

Sleep loss can be conceptualized a physiological stressor, withboth subjective (psychological) and physiological effects(described further below). The multiple systems involved in thephysiologic stress response are homeostatic and tightly inter-related (Almawi et al., 1996; de Kloet, 2000) and include thesympatho-adrenal, the hypothalamic-pituitary-adrenal (HPA), aswell as the inflammatory system. Inflammatory cytokines serveas chemical messengers and are negatively controlled by cortisol,a glucocorticoid (GC) that is the main output hormone of theHPA axis (reviewed in Chrousos, 2009). Impaired GC sensitivityhas been reported in response to various acute and chronic stres-sors (Herman et al., 1995; Miller et al., 2002; Stark et al., 2001),and GC sensitivity is one possible mechanism by which observedincreases in inflammatory markers can be explained.

The HPA system is perhaps the most studied stress responsesystem, and is known to typically habituate when faced withrepeated or ongoing stressors (Grissom and Bhatnagar, 2009).

mmun.

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2 N.S. Simpson et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx

However, sleep loss is a unique stressor because it is a biologicalresource necessary for regulation of multiple physiological sys-tems, including the stress response system (Hamilton et al.,2007; McEwen, 2006). Further, in extreme cases, sleep is necessaryfor survival itself (Everson et al., 1989; Montagna et al., 1995). Noprior research has examined whether humans can adapt to chronicpatterns of insufficient sleep and limited recovery, or studied theimpact of this common real-world pattern on stress-response sys-tems. As described below, the impact of single episodes of sleeploss and (to a lesser extent) recovery sleep has been tested, how-ever it remains unknown whether these results remain true whenpatterns of restricted sleep and recovery become chronic.

Within single episodes of experimental sleep loss, subjectiveratings of sleepiness, positive mood, and self-reported physicalfunctioning appear to show response stabilization, or acclimation.For example, subjective experiences of pain (Haack andMullington, 2005) and sleepiness (Van Dongen et al., 2003) stabi-lize after a few days of sleep restriction or sleep deprivation (ordeteriorate more slowly), despite ongoing sleep loss. On a physio-logical level, multiple markers of the stress system have beenfound to increase following a single episode of sleep loss, includingcortisol (Balbo et al., 2009; Guyon et al., 2014) and the inflamma-tory marker interleukin [IL]-6 (Haack et al., 2007; Irwin et al.,2006; Pejovic et al., 2013; van Leeuwen et al., 2009; Vgontzaset al., 2004). Although habituation to acute stressors is a key fea-ture of the HPA system (Grissom and Bhatnagar, 2009), it isunknown whether this classic pattern of habituation can beapplied to the physiological stress of repeated exposures to sleeploss with limited recovery sleep, given that sleep loss is a uniquephysiological stressor.

Little is known about the impact of repeated episodes of sleeploss or the role of recovery sleep. To our knowledge, the currentstudy protocol tests the longest model of chronic sleep restrictionto date. Everson and colleagues have conducted studies of repeatedexposure to sleep loss and recovery in an animal model, and havedocumented changes in metabolic indices (weight, food intake),and pathological organ and bone changes (Everson and Szabo,2009, 2011). Recovery from sleep loss has been rarely studied,but using a five night sleep restriction/two night recovery protocol,van Leeuwen and colleagues showed that IL-6 mRNA levelsremained elevated after two nights of recovery sleep (vanLeeuwen et al., 2009). These data provide preliminary support that‘catching-up’ on sleep over the weekend might be insufficient torestore stress-response systems, and contribute to ongoingresponses to repeated exposure to sleep loss over time. These lim-ited data highlight a critical gap in our understanding of conse-quences of insufficient sleep, as it is the real-world experiencesof repeated episodes of sleep loss and limited recovery sleep thatare most likely to have a long term impact on health.

This study modeled real-world sleep-wake patterns of sleeprestriction and recovery in the laboratory environment to investi-gate effects on multiple stress systemmarkers, using an intensifiedmodel of sleep restricted to four hours of sleep on weekdays andextended to eight hours on weekends. This amplification of themagnitude of difference between weekdays/weekends was chosenin part due to the aim of assessing the impact of these patternsunder highly controlled experimental conditions that can be main-tained for a period of weeks, rather than the months or years thatadults often will maintain these milder patterns of sleep restrictionand recovery in the real world.

Based on previous research, we hypothesized that there wouldbe a response stabilization or habituation across repeated episodesof sleep loss in subjective domains, but poor habituation and anincomplete recovery in physiological domains. If true, these find-ings could help explain why patterns of inadequate sleep persist,namely, because there would be no perceived negative impact of

Please cite this article in press as: Simpson, N.S., et al. Repeating patterns of s(2016), http://dx.doi.org/10.1016/j.bbi.2016.06.001

these behavior patterns. Additionally, this study was specificallydesigned to extend previous research demonstrating that sleep lossresults in increases in serum or plasma IL-6 (Haack et al., 2007;Irwin et al., 2006; Pejovic et al., 2013; van Leeuwen et al., 2009;Vgontzas et al., 2004) by focusing on monocytes, and whetherthe expected increased expression of inflammatory mediatorscan be explained by changes in the sensitivity of monocytes tocortisol.

2. Methods

2.1. Experimental model

The hypothesis was tested using a sleep restriction conditionconsisting of three weeks of a repeating pattern of five nights ofsleep restricted to 4 h/night (0300–0700 h) followed by two nightsof recovery sleep with 8 h/night (2300–0700 h). This model wasdesigned to mirror commonly observed patterns of moderatelyrestricting on weeknights and recovering sleep on weekend nightsthat often occur in the general population for periods of months oryears (National Sleep Foundation, 2010), albeit with an amplifiedsleep restriction pattern on weeknights (see Fig. 1). This amplifiedsleep restriction period was designed to maximize the potentialthat the effects of what are often much longer periods of mildersleep restriction and recovery that occur in the real world couldbe captured in a relatively short three-week in-laboratory experi-mental protocol. The sleep control condition consisted of threeweeks with a nightly sleep opportunity of 8 h. In an intra-individual randomized balanced design, participants underwenttwo 25-day in-hospital stays (restricted sleep condition and sleepcontrol condition) separated by more than two months. Each 25-day stay started with an adaptation and a baseline day, followedby three weeks of either the repeated exposure to sleep restric-tion/recovery or control sleep, and ended with an additional nightof full sleep (totaling 25 days).

2.2. Participants

This study was approved by the Institutional Review Board forthe Protection of Human Subjects at the Beth Israel DeaconessMedical Center (BIDMC). Participants were recruited via commu-nity advertisements. Seventeen healthy young women and menwere studied. Fourteen participants completed both 25-day-in-hospital conditions; three participants could only complete oneof the two 25-day-in-hospital conditions due to change in work/family-related requirements (see Fig. 2).

Participants were between the ages of 18–35 years, had a bodymass index (BMI) between 18.5 and 30 kg/m2, a daily sleep dura-tion between 7 and 9 h (verified by sleep diary data over a 2-week period), began their habitual sleep period within one hourof 11 pm (to ensure normal entrainment) and had blood chemistrylevels within the normal range. Female participants were eligible ifthey had regular menstrual cycles and no significant discomfortduring pre-menses/menses. Exclusion criteria included presenceor past history of major medical problems, psychiatric disordersor sleep disorders. Additional exclusion criterion included preg-nant/nursing status, regular medication use other than oral contra-ceptives, and donation of blood or platelets three month prior to orin-between study stays.

2.3. Study protocol

2.3.1. Screening & randomizationParticipants were initially screened over two visits to the hospi-

tal and were evaluated by a study physician for the exclusion

leep restriction and recovery: Do we get used to it?. Brain Behav. Immun.

http://dx.doi.org/10.1016/j.bbi.2016.06.001

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Fig. 1. Study protocol: Repeated exposure to sleep restriction-recovery patterns. In the control condition, participants had a sleep opportunity of 8 h every day.

N.S. Simpson et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx 3

criteria described above. Two weeks before entering each 25-dayin-hospital stay, participants were asked to follow the study sleepschedule (11 pm–7 am), which was verified by sleep log data. Theweek prior to the second 25-day visit, blood tests used at the initialscreening were repeated to ensure values are in the normal range.Participants were randomized to the order of experimental condi-tions (sleep restriction or control) on the first day of the first 25-day hospital stay. An independent statistician prepared envelopeswith randomization codes, one of which was opened by a seniorstaff member prior the first hospital stay.

2.3.2. Research environmentDuring the two 25-day in-hospital stays (Fig. 1), participants

stayed in a private or semi-private room in the Clinical ResearchCenter (CRC) at BIDMC. Intensive physiological recordings wereconducted on seven out of the 25 days: on the baseline night, everyfifth day of restricted/control sleep and every second night ofrecovery/control sleep in each of the three weeks. These intensivemeasurement periods included PSG recordings and frequent bloodsampling though an intravenous catheter across 24 h. Subjectivewell-being assessments were also collected on computerizedvisual analog scales every four hours throughout waking periods.

During the sleep restriction nights, the sleep opportunity wasfrom 0300 to 0700 h; however, participants had to remain in bedin a semi-supine position during the wakeful nighttime periods(2300–0300 h) in order to limit differences in postural and phys-ical activity inputs across all study nights and conditions. Lightlevels were less than 40 lx during wakeful nighttime periods(2300–0300 h). During daytime periods (0700–2300 h), partici-pants had access to both artificial and natural light sources.Throughout both 25-day stays, participants were maintained ona balanced diet (NA+ and K+ controlled) and regimented fluidintake in order to maintain body weight/composition throughoutthe study. Meals and fluids were served at standardized hours. Toprevent sedentary conditions and maintain constant activitylevels, participants had scheduled walks (5–10 min each) withinthe CRC every other hour throughout the daytime periods ofthe protocol (between 0700 and 2300 h). On non-intensiverecording days, participants had an additional longer walk ofapproximately 30 min that could take place outdoors. Participantswere also encouraged to follow their pre-study exercise habits


through an opportunity on the non-intensive recording days tovisit the hospital gym facilities. Room temperature was adjustedto each participant’s comfort level during the first two adaptationdays, and the same daytime temperature was kept throughoutthe remaining days of the protocol. Nighttime temperature(2300–0700 h) was set 2 �C lower than daytime temperature. Par-ticipants were allowed to have visitors during daytime periods, aswell as have access to email and phone, in order to minimize dis-ruptions to their social networks and prevent social isolation.During all waking periods a research assistant accompanied par-ticipants in order to ensure adherence to the study protocol andprocedures, as well as to engage participants in social activitiessuch as board/video games or talking, as needed.

2.3.3. Measurements2.3.3.1. Polysomnographic recording (PSG). Sleep was recorded usingthe Embla system N7000 (Medcare US, Buffalo) on seven intensiverecording days of each 25-day study run (at baseline, every fifthand seventh day of each of the three weeks). The PSG montage fol-lowed standard criteria and sleep electroencephalography wasmanually stage-scored on a 30 s epoch basis (American Academyof Sleep Medicine, 2007). All recordings were scored by the samesleep technician.

2.3.3.2. Blood sampling. On the seven intensive recording days ofeach 25-day study run, blood was drawn at 2-hourly intervalsusing an indwelling 20-gauge forearm catheter. During sleepopportunities, a long line was attached to the catheter and bloodcollection was performed from an adjacent anti-chamber withoutdisrupting the participant’s sleep. The total amount of blood takenover each 25-day protocol did not exceed 550 ml.

2.4. Stress response system measures

2.4.1. CortisolCortisol was measured in serum collected every two hours on

the seven intensive recording days using an electrochemilumines-cence immunoassay (ECLIA, Labcorp.com). According to thecompany, intra-run and inter-run precision are 1.2% and 1.6%,respectively.



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Fig. 2. Ratings of ‘Sleepy’, ‘Effort to do anything’, and ‘Stressed’ across therepeated exposure to sleep restriction-recovery patterns. Data present estimatedmean ± SEM based on mixed model analysis. *p < 0.05 between conditions.



2.4.2. Stimulated IL-6Stimulated IL-6 was measured in vitro as the capacity of mono-

cytes to express IL-6, using the 1130 h blood sample on each of theseven intensive recording days. Whole blood was stimulated withlipopolysaccharide (LPS) from Escherichia coli 0127-B8 (LPS 100 pg/ml, Sigma-Aldrich), and then brefeldin A (10 lg/ml, Sigma-Aldrich)was added to the sample, which was incubated for 4 h at 37 �C in a5% CO2 atmosphere. Following fixation and permeabilization pro-cedures (IntraprepTM Permeabilization reagents [Beckman Coulter]),fluorescence-conjugated antibodies were added (CD14 APC, CD45KrO [both Beckman Coulter], IL-6 PE [BD Bioscience]) and samplesincubated for 15 min at room temperature in the dark. Sampleswere vortexed, washed with phosphate-buffered saline solution(PBS 1X, Sigma Aldrich), and stored at 2–8 �C in the dark after re-suspension in 500 ul of PBS containing 0.5% formaldehyde. Prepa-rations were analyzed within 24 h using a GalliosTM flow cytometer(Beckmann-Coulter) at the Flow Cytometry Core at BIDMC, and100,000 events were acquired. Percentage of IL6-positive mono-cytes (LPS-stimulated and spontaneous) were quantified usingKaluza� Flow Analysis software (Beckmann Coulter).

2.4.3. Unstimulated IL-6The same procedures were applied to a whole blood sample

that was not stimulated with LPS.

2.4.4. Glucocorticoid (GC) sensitivity of monocytesGlucocorticoid (GC) sensitivity of monocytes was determined

by the capacity of the synthetic glucocorticoid dexamethasone(DEX) to suppress IL-6 expression in monocytes, using the 1130 hblood sample on each of the seven intensive recordings days.Whole blood was stimulated with LPS (see above), and then differ-ent concentrations of DEX (25, 50, 100, 200, and 400 nM; Sigma-Aldrich) as well as brefeldin A were added to the samples, whichthen underwent the same procedures as described above. For sta-tistical purposes, IL-6 suppression curves were calculated aschange from monocytic IL-6 expression without DEX. In addition,area under the curve (AUC) was computed for each IL-6 suppres-sion curve according to methods described by Pruessner and col-leagues (Pruessner et al., 2003). For this analysis, samples withdifferent DEX concentrations were first calculated as change frombaseline (i.e., sample without DEX), and then computed as AUC.

2.4.5. Subjective measuresSubjective measures were assessed every four hours during the

waking periods of the protocol. Participants were asked to rate theintensity of various well-being items using computerized visualanalogue scales (AsWin, programmed by Martin Rivers & Associ-ates). The VAS set used in the current study contained items fromthe Deactivation Activation Check List (Thayer, 1978) and scaleshave been used in our previous studies (Haack and Mullington,2005; Haack et al., 2009). The test battery required approximatelyfive minutes per administration. Ratings of ‘Sleepy’, ‘Effort to doanything’, and ‘Stressed’ were aggregated across the daytime peri-ods of each study day for statistical analysis.

2.5. Statistics

Linear mixed models were used to analyze the data, with condi-tion (repeated sleep restriction vs. control sleep) and study day(baseline, fifth and seventh day of each of the three weeks) as fixedfactors, and participants and participants � day as random factors.For variables that were also repeated within a study day (e.g.,cortisol measured every two hours, IL-6 suppression measured atvarious concentrations of DEX at each recording day), time ofday/concentration were also entered as additional fixed factors.The baseline day was used as a covariate in order to account for




differences at study start. Accordingly, data in graphs are depictedas estimated means from mixed model analysis. Since baseline daywas used as covariate, interpretation of the interaction as well asmain condition effects is considered appropriate and are presentedif significant. Physiological stress outcome measures were: (1)serum cortisol assessed every two hours during intensive record-ing days, (2) IL-6 positive monocytes (LPS-stimulated and unstim-ulated), assessed once per intensive recording day at 11:30 h, and(3) GC sensitivity of monocytes, measured as IL-6 suppressioncurves at various doses of DEX once per intensive recording dayat 11:30 h, and calculated as AUC (Pruessner et al., 2003). Subjec-tive outcome measures were ratings of ‘Sleepy’, ‘Effort to do any-thing’, and ‘Stressed’, which were aggregated to a single daytimemean (0700–2300) across each of the seven intensive recordingdays.

3. Results

Of the 17 participants randomized, 14 completed both sleeprestriction and sleep-control laboratory stays. Table 1 presentsbaseline characteristics of the participants who were randomizedand are included in analyses. On average, there were144 ± 23 days (4.8 ± 0.8 months) between laboratory stays. Dataand statistical analyses are described below; Supplemental Table 1presents summary data from subjective and physiological stressmarkers across repeated patterns of sleep restriction andrecovery.

3.1. Subjective well-being responses

Fig. 2 presents the subjective well-being responses to therepeated exposure of sleep restriction-recovery patterns. Mixedmodel revealed a significant condition effect (p < 0.05) for rat-ings of ‘Sleepy’ and ‘Effort to do anything’, but not for ‘Stressed’.Values for ‘Sleepy’ and ‘Effort to do anything’ significantlyincreased during the sleep restriction days of each week, andalmost completely returned to baseline values during intermit-tent recovery sleep nights (no significant difference comparedto baseline). When comparing ratings of ‘Sleepy’ across consec-utive weeks in the sleep restriction group, mixed model analy-ses indicated a significant week effect. Ratings wereprogressively lower from week to week, indicating that the sub-jective experience of feeling sleepy habituated across therepeated exposure of sleep restriction-recovery patterns. Simi-larly, ratings of ‘Effort to do anything’ trended towards a signif-icant week effect (p < 0.07), indicating that the subjectiveexperience of ‘Effort to do anything’ somewhat habituates tothe repeated exposure of sleep restriction-recovery patterns.Ratings of ‘Stressed’ did not show any significant increasesbetween sleep restriction periods.

Table 1Participant characteristics.

Controlsleep

Restrictedsleep

N* 16 15Sex Female/Male 8/8 7/8Age (yrs) Mean ± SEM 24.9 ± 1.1 24.9 ± 1.2Screening BMI (kg/m2) Mean ± SEM 24.8 ± 0.8 24.6 ± 0.7Habitual sleep duration (h)** Mean ± SEM 8.1 ± 0.2 8.4 ± 0.1

* 14 of the participants completed both 25-day stays (control sleep and restrictedsleep; 3 participants completed only 1 stay.** Based on 10–14 day recording period through diary.


3.2. Physiological stress responses

3.2.1. Diurnal cortisol rhythmFig. 3 presents daily serum cortisol levels across the repeated

exposure to sleep restriction and recovery patterns. Mixed modelanalysis indicated a significant interaction effect between condi-tion by day by time of day. As seen in Fig. 3, cortisol levels areincreasingly dysregulated across repeated exposure to sleeprestriction, as indicated by the increasing number of significanttime point differences in Fig. 3. Most consistently, fasted cortisollevels shortly after awakening (0730) are increasingly higher dur-ing the repeated sleep restriction exposure compared to controlsleep participants, as indicated by a significant week effect inmixed model analysis (Fig. 4). Though not significantly different,morning cortisol levels did not completely return to baseline levelsafter two nights with an 8 h sleep opportunity (see Fig. 4).

3.2.2. IL-6 positive monocytesFig. 5a presents the percentage of LPS-stimulated IL-6 positive

monocytes throughout the repeated exposure to sleep restrictionand recovery. Mixed model analysis indicated a significant condi-tion effect. Compared to the control sleep condition, values weresignificantly higher during the second and third week of sleeprestriction. Values remained higher after two nights of recoverysleep following the second week (p < 0.05) and third week(p = 0.09) of restricted sleep. Fig. 5b presents percentage of non-stimulated IL-6 positive monocytes. Mixed model analysis indi-cated a significant condition effect. When compared to the controlsleep condition, non-stimulated IL-6 levels were significantlyhigher during the first sleep restriction week (p < 0.05) and trendedto be higher during the second and third sleep restriction week(both p = 0.06). Levels stayed significantly higher after two nightsof recovery sleep following the first sleep restriction exposure(p < 0.05) and trended to be higher after the second sleep restric-tion exposure (p = 0.07).

3.2.3. Glucocorticoid (GC) sensitivity of monocytesFig. 6 presents the GC sensitivity determined by the ability of

dexamethasone (DEX) to suppress IL-6 expression in monocytesacross repeated exposure to sleep restriction-recovery patterns.Mixed model analysis indicated a significant interaction effectbetween condition and day. While GC sensitivity was not signifi-cantly affected during the first sleep restriction exposure, it wassignificantly higher during the second and third sleep restrictionexposures when compared to control sleep. GC sensitivity trendedto remain higher after two nights of recovery sleep following thethird exposure to restricted sleep (p < 0.06). Fig. 7 depicts the areaunder the curve (AUC) for the IL-6 suppression curves. Mixedmodel analyses revealed a significant condition effect, due tohigher GC sensitivity throughout the three weeks of sleeprestriction-recovery exposure, when compared to control sleep.

4. Discussion

This study, to the best of our knowledge, provides the first evi-dence for the impact of real-world sleep patterns of sleep restric-tion and recovery on stress response systems. Consistent withour hypotheses, repeated episodes of restricted sleep and recoverywere not experienced as subjectively stressful. While they wereperceived more generally as ‘burdensome’ (as reflected by increas-ing subjective symptoms of sleepiness and effort), participants’subjective responses acclimated to repeated exposure to sleeprestriction and tended to fully recover after two nights of full sleep.In contrast, the physiological stress response systems assessed(cortisol and inflammatory) showed increased activity and did



Week 1 5th day of sleep restriction 2nd day of recovery sleepBaseline

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Fig. 3. Diurnal cortisol rhythms across the repeated exposure sleep restriction-recovery patterns. Data present mean ± SEM. *p < 0.05 between conditions.


not habituate or fully recover when repeatedly exposed to thissleep restriction-recovery pattern. Further, the observed increasedGC sensitivity of monocytes suggests that there was a disruptedinterplay between the HPA and inflammatory system. The fact thatthese escalating physiological responses were dissociated fromsubjective impact suggests one reason that these behavior patternspersist despite accumulating physiological costs. Plainly stated, ifthe person does not ‘feel’ an accumulated negative impact of thesesleep patterns, there is no internal motivation to change thebehavior.

The current study furthers previous research that experimen-tally modeled ‘catching up’ after a single week of sleep restrictionis insufficient to restore the homeostasis of the inflammatoryresponse system (van Leeuwen et al., 2009) by demonstrating thatwhen repeatedly exposed to such sleep restriction-recovery peri-ods, LPS-stimulated IL-6 positive monocytes increase and do notappear to habituate to the repeated exposure to sleep restriction,and two nights of recovery sleep do not normalize levels. IL-6expression in unstimulated monocytes were also significantly ele-vated during the first week of sleep restriction followed by sleep


recovery (paralleling Irwin et al., 2015b), and tended to remain ele-vated while these sleep patterns continue. These findings indicatethat even in the absence of an exogenous activation of innateimmune components (e.g., LPS), monocytes spontaneously pro-duce more IL-6 in response to sleep loss, do not habituate withthe repeated exposure to sleep loss, and do not fully recover evenafter a limited opportunity for recovery sleep.

These findings contrast, at least to some extent, to the recentmeta-analytic finding that sleep disruption, rather than habituallyshort sleep durations and experimentally modeled sleep loss arenot associated with increased IL-6 (Irwin et al., 2015a). However,this meta-analysis also demonstrates the variability in IL-6 find-ings, which is likely introduced, at least in part, by the level ofexperimental control in each study, including as the impact of foodcomposition, timing of meal intake relative to blood draws, andwhether the participants were resting quietly in a seated periodprior to blood sample collection. Perhaps more importantly, thereported IL-6 results may be more closely tied to the range of mag-nitudes and durations of sleep loss examined across studies. Thecurrent study is the first that closely models the chronicity of



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Fig. 4. Cortisol levels after awakening across repeated sleep restriction-recoverypatterns. Data present estimated mean ± SEM based on mixed model analysis.*p < 0.05 between conditions.

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Fig. 5. IL-6 positive monocytes assessed in (a) LPS-stimulated and (b) unstimulatedwhole blood across repeated exposure to sleep restriction-recovery patterns. Datapresent estimated mean ± SEM based on mixed model analysis. *p < 0.05 betweenconditions.


real-world patterns of sleep loss, is designed to take a more mech-anistic approach by investigating whether changes in the sensitiv-ity of monocytes to the counter-inflammatory signal cortisol maybe responsible for increased IL-6 expression, and is highly con-trolled, leaving little room for confounds from experimentalfactors.

Sleep loss appears to be a somewhat unique physical stressor, inthat the HPA response to sleep loss compared to other stressors ismild (Balbo et al., 2010; Guyon et al., 2014; Meerlo et al., 2008).Additionally results from this study demonstrates that chronicsleep loss does not produce the typical pattern of habituation withrepeat exposure with respect to HPA (Grissom and Bhatnagar,2009) and IL-6 responses (the latter when measured on a cellularlevel (McInnis et al., 2015)). While it is adaptive for the HPA axisto habituate to non-harmful stressors, sleep is a necessary biolog-ical resource (Everson and Szabo, 2009; Hamilton et al., 2007;McEwen, 2006), so habituation to chronic sleep loss may be harm-ful rather than adaptive. While the changes in HPA axis functioningobserved in the current study are small, as they have been in pre-vious studies of experimental sleep loss, there may be a cumulativeeffect after months and potentially years of insufficient sleep. Addi-tionally, there is increasing evidence that small changes in inflam-matory and stress mediators are present in a variety of diseases,including cardiovascular, metabolic, neurodegenerative diseases,as well as some forms of cancer and pain conditions, which pro-vides further support for the importance of the small changesobserved in the current study (Medzhitov, 2010).

One possible explanation for the observed increase IL-6 is thatIL-6 producing monocytes became less sensitive to the counter-inflammatory signal of cortisol (i.e., glucocorticoid sensitivitydecreased). Stress-induced activation of the HPA and inflammatorysystems is metabolically costly, with potential deleterious effects ifthese systems are overactive. Therefore, while it is adaptive for theHPA axis to habituate to non-harmful stressors, in this context itmay be harmful to adjust to the stress of chronic sleep loss giventhat sleep is a necessary biological resource (Everson and Szabo,


2009) and, more globally, sleep is thought to be required to ade-quately adapt to a stressor (Hamilton et al., 2007; McEwen,2006). The process of habituation to repeated stress is, in part, reg-ulated by cortisol negative feedback mechanisms, as demonstratedby inhibited habituation with blockage of the GC receptor(reviewed in Grissom and Bhatnagar, 2009). HPA and inflammatorysystems are tightly regulated, and the GC cortisol is crucial for theappropriate termination of every stress response via inhibition ofmonocytes and other immune cell populations in the productionof transcription factors (such as NF-kB) and downstream inflam-matory cytokines, such as IL-6. However, in parallel to this IL-6increase, we also observed an increase in GC sensitivity; one thatdid not appear to be sufficient to prevent IL-6 production by mono-cytes. Previous research has found contrasting results wheredecreased GC sensitivity is observed (along with increasing inflam-mation) under condition of chronic stress (e.g., Cohen et al., 2012);it is challenging to expand upon the discussion of how the mecha-nisms differ between those studies and ours without additionalresearch. However this phenomenon of increased IL-6 productiondespite increased GC sensitivity observed in the current study




DEX concentration (nM) 0 25 50 100 200 400



Week 1

Week 2

Week 3

5th day of sleep restriction 2nd day of recovery sleep Baseline

5th day of sleep restriction 2nd day of recovery sleep

5th day of sleep restriction 2nd day of recovery sleep

0 25 50 100 200 400 0 25 50 100 200 400

0 25 50 100 200 400 0 25 50 100 200 400

P

* *

* *

* *


IL-6

sup

pres

sion

by

dexa

met

haso

ne (A

UC

)G

C s

ensi

tivity

Lower

Higher-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

3 8 10 15 17 22 24

Day

Repeated exposure to restricted sleepControl sleep

Fig. 7. GC sensitivity calculated as AUC of IL-6 suppression. Higher IL-6 suppressionby DEX indicates higher GC sensitivity. Data present estimated mean ± SEM basedon mixed model analysis.*p < 0.05 between conditions.


during the week and eight hours on weekends) was utilized. Theextent to which patterns of stress responses will change if milderpatterns of restricted sleep and recovery were carried out for alonger period of time, (e.g., years or decades), as are often experi-enced in real life, will need to be addressed in future studies.

5. Conclusion

To our knowledge, this is the first study that has used an in-laboratory design to model patterns of repeated sleep restrictionand recovery that are prevalent in modern society. It is also amongthe first that begins to map out a mechanistic path of multiplestress responses systems in the context of experimental sleep lossin humans. Despite habituation in subjective domains, weobserved that physiological stress systems show patterns of con-tinued elevated responses across repeated cycles of sleep restric-tion, even with limited opportunities for recovery sleep. Thecurrent study provides preliminary, yet powerful evidence thatwe cannot fully adjust to patterns of restricted sleep loss andrecovery. Despite accumulating physiological impact, if the subjec-tive experience to these sleep patterns is one of habituation, it caneasily be seen why obtaining insufficient sleep on a chronic basis isexperienced as benign and why motivation to change these behav-ior patterns remains low. The growing awareness of chronic low-grade inflammation as a basis for increasing rates of cardiovascu-lar, metabolic, pain or mood related disorders (Medzhitov, 2010)suggests that these patterns of insufficient sleep may pose a signif-icant health risk. Given its high prevalence in modern society, theimpact of these patterns of chronically restricted sleep with limitedrecovery on long-term health cannot be ignored.

Acknowledgments

This work was funded by Grants R01 HL 105544 from theNational Heart, Lung, and Blood Institute, and Grants UL1RR02758 and M01-RR-01032 from the National Center for


Research Resources to the Harvard Clinical and TranslationalScience Center.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbi.2016.06.001.

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Repeating patterns of sleep restriction and recovery: Do we get used �to it?1 Introduction2 Methods2.1 Experimental model2.2 Participants2.3 Study protocol2.3.1 Screening & randomization2.3.2 Research environment2.3.3 Measurements2.3.3.1 Polysomnographic recording (PSG)2.3.3.2 Blood sampling

2.4 Stress response system measures2.4.1 Cortisol2.4.2 Stimulated IL-62.4.3 Unstimulated IL-62.4.4 Glucocorticoid (GC) sensitivity of monocytes2.4.5 Subjective measures

2.5 Statistics

3 Results3.1 Subjective well-being responses3.2 Physiological stress responses3.2.1 Diurnal cortisol rhythm3.2.2 IL-6 positive monocytes3.2.3 Glucocorticoid (GC) sensitivity of monocytes

4 Discussion5 ConclusionAcknowledgmentsAppendix A Supplementary dataReferences

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Repeating patterns of sleep restriction and recovery: Do ...€¦ · Full-length Article Repeating patterns of sleep restriction and recovery: Do we get used to it? Norah S. Simpsona,