Assessment of the cortisol awakening response : Expert consensus guidelines

Assessment of the cortisol awakening response: Expert consensus guidelines

Tobias Stalder

, Clemens Kirschbaum

, Brigitte M. Kudielka

, Emma K. Adam

, Jens C. Pruessner

, Stefan Wüst

, Samantha Dockray

, Nina Smyth

, Phil Evans

,

Dirk H. Hellhammer

, Robert Miller

, Mark A. Wetherell

, Sonia J. Lupien

, Angela Clow

aDepartmentofPsychology,TUDresden,Dresden,Germany

bDepartmentofPsychology,UniversityofRegensburg,Regensburg,Germany

cSchoolofEducationandSocialPolicy,NorthwesternUniversity,Evanston,USA

dDepartmentofPsychiatry,McGillUniversity,Montreal,Canada

eSchoolofAppliedPsychology,UniversityCollegeCork,Cork,Ireland

fDepartmentofPsychology,UniversityofWestminster,London,UK

gDepartmentofPsychology,TrierUniversity,andStresszentrumTrier,Germany

hDepartmentofPsychology,NorthumbriaUniversity,Newcastle,UK

iDepartmentofPsychiatry,UniversityofMontreal,Montreal,Quebec,Canada

Keywords:

CAR Measurement Saliva Adherence Covariates Guidelines

a b s t r a c t

Thecortisolawakeningresponse(CAR),themarkedincreaseincortisolsecretionoverthefirst30–45min aftermorningawakening,hasbeenrelatedtoawiderangeofpsychosocial,physicalandmentalhealth parameters,makingitakeyvariableforpsychoneuroendocrinologicalresearch.TheCARistypically assessedfromself-collectionofsalivasampleswithinthedomesticsetting.Whilethisconfersecological validity,itlacksdirectresearcheroversightwhichcanbeproblematicasthevalidityofCARmeasure- mentcriticallyreliesonparticipantscloselyfollowingatimedsamplingschedule,beginningwiththe momentofawakening.ResearchersassessingtheCARthusneedtotakeimportantstepstomaximizeand monitorsalivasamplingaccuracyaswellasconsiderarangeofotherrelevantmethodologicalfactors.

Topromotebestpracticeoffutureresearchinthisfield,theInternationalSocietyofPsychoneuroen- docrinologyinitiatedanexpertpanelchargedwith(i)summarizingrelevantevidenceandcollective experienceonmethodologicalfactorsaffectingCARassessmentand(ii)formulatingclearconsensus guidelinesforfutureresearch.Thepresentreportsummarizestheresultsofthisundertaking.Consensus guidelinesarepresentedoncentralaspectsofCARassessment,includingobjectivecontrolofsampling accuracy/adherence,participantinstructions,covariateaccounting,samplingprotocols,quantification strategiesaswellasreportingandinterpretingofCARdata.Meetingthesemethodologicalstandardsin futureresearchwillcreatemorepowerfulresearchdesigns,thusyieldingmorereliableandreproducible resultsandhelpingtofurtheradvanceunderstandinginthisevolvingfieldofresearch.

Contents

1. Introduction...415

2. Cortisolawakeningresponse...416

2.1. Descriptionanddistinctivefeatures...416

2.2. Maincomponents...416

3. Inaccuratesampling:prevalenceandimpact...417

∗ Correspondingauthorat:TechnischeUniversitätDresden,DepartmentofPsy- chology,ZellescherWeg19,01069Dresden,Germany.Fax:+4935146337274.

E-mailaddresses:tobias.stalder@tu-dresden.de,tobias.stalder@googlemail.com (T.Stalder).

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-x4dttsqcxssg4 Erschienen in: Psychoneuroendocrinology ; 63 (2016). - S. 414-432

https://dx.doi.org/10.1016/j.psyneuen.2015.10.010

3.1. Delaybetweenawakeningandinitiationofsampling...417

3.2. Inaccuratepost-awakeningsampling...419

4. Strategiesfordealingwithinaccuratesampling...419

4.1. Objectivemonitoringstrategies...419

4.1.1. Methodsforverifyingawakeningandsamplingtimes...419

4.1.2. Dealingwithverifiedinaccuratedata...420

4.2. Arethereviablestrategieswithouttheuseofobjectivemeasures?...420

4.2.1. Forcedawakening...420

4.2.2. Increasingstatisticalpower...421

4.2.3. ExclusionofCARnon-responders...421

4.2.4. Informationalstrategies...421

5. Maximizingadherence...422

6. Dealingwithcovariates...423

6.1. Instructionsaboutpost-awakeningbehavior...423

6.2. Controlvariables...424

6.2.1. Statecovariates...424

6.2.2. Traitcovariates...425

6.3. Exclusioncriteria...425

7. Proceduralanddesignconsiderations...426

7.1. Samplingtimes...426

7.2. Numberofstudydays(cross-sectionalresearch)...426

7.3. Samplestorageandcortisolanalysis...426

7.4. Statisticalconsiderations...427

8. Summaryandguidelines...427

Conflictofinterest ... 429

Consensusprocessandcontributions ... 429

Roleofthefundingsource...429

Acknowledgement...429

References...429

1. Introduction

Abnormalsecretionoftheglucocorticoidhormonecortisolas the final product of the hypothalamus-pituitary-adrenal (HPA) axis is considered a crucial factor in linking the experience of chronicpsychosocialstresstoadverseeffectsonhealth(Chrousos, 2009).Besidesreactivitytoacutestressors,changestothecirca- dianregulationofcortisolsecretionareconsideredimportantin thiscontext(KondratovaandKondratov,2012;MenetandRosbash, 2011;Naderetal.,2010).Anaspectofcortisolregulationthatis ofspecialinteresttopsychoneuroendocrinological(PNE)inquiry is thecortisol awakening response (CAR), which describesthe markedincreaseincortisollevelsacrossthefirst30–45minfol- lowingmorningawakening(Clowetal.,2004,2010;Elderetal., 2014;KudielkaandWüst,2010).TheCARwasfirstsystematically describedinthemid-1990s(Pruessneretal.,1997)andsoongained attentionasa favorable biomarkerinPNE researchduetosev- eralmethodologicaladvantagesoverpreviouslyemployedcortisol assessmentstrategies(seeSection2).Theseadvantagestogether withevidenceshowinguniqueassociationsoftheCARwithpsy- chosocial,psychiatricandhealth-relatedparametershaveresulted in a rapid increase in publications over the past15 years (see Fig.1a).

TheCARcombinesfeaturesofareactivityindex(responseto awakening)withaspects tiedtocircadian regulation(occurring roughly at the same time every 24h) making it a fascinating researchtopic.However,preciselythesefeaturesalsomakeaccu- rateassessmentoftheCARachallengingtask.WhenrelyingonCAR dataacquiredbyparticipantsthemselves(usuallythroughsaliva sampling),validitycriticallyreliesonparticipantscloselyfollowing atimedsamplingschedule,beginningwiththemomentofawaken- ing.Inaccuratesampletimingcanoccureasilyandcansubstantially biasCARestimates.Furthermore,anumberofothermethodolog- ical factors,such as accounting for covariates,the number and natureofstudydaysand thetimingofsampling,canmarkedly affectCARdata.Whilenotallquestionsregardingtheroleandreg-

99-00 01-02 03-04 05-06 07-08 09-10 11-12 13-14

snoitacilbuP

0 50 100 150 200

(b) CAR research between 2013 and 2014 – methodological aspects^b Assessment days (mean, range)^c: 2.0, 1–5

1 day: 31.7% 2 days: 47.1% 3 days: 16.3% 4–5 days: 4.8%

Sampling mes during first 1h (mean,range): 2.7, 1–7

1 sample: 3.6% 2 samp.: 51.1% 3 samp.: 18.7% 4–5 samp.: 25.9%

Reported use of diary log method: Yes: 65.5% No: 34.5%

Objecve controlof awakening me^d: Yes: 7.9% No: 92.1%

Objecve controlof sampling mes^e: Yes: 18.6% No: 81.4%

Full objecve control (both methods)^f: Yes: 5.7% No: 94.3%

(a) Publicaon count of research using CAR assessments over the past 15 years^a

Fig.1.(a)DevelopmentofpublicationsusingCARassessmentsoverthepast15 yearsand(b)methodologicalaspectsofresearchfromthelastperiodbetween2013 and2014.

ulationoftheCARhavebeenadequatelysolveduntilnow,several carefulinvestigationshaveexaminedtheimpactofmethodolog- icalfactorsonaccurateCARassessmentandhaverecommended strategiesfordealingwiththem(describedbelow).Unfortunately, suchrecommendationshavenotbeenwidelyimplementedinpub- lishedCARresearch.Fig.1bprovidesanoverviewofmethodological characteristicsofsuchstudies,publishedbetween2013and2014.

Itcanbeseenthattheemployedmethodologicalstandardsvar- iedwidelybetweeninvestigations,withahighnumberofstudies fallingshortofpreviousrecommendationsforbestpracticeinCAR research(e.g.,objectivecontrolofsamplingtimes:Brodericketal., 2004;Kudielkaetal.,2003).

Toaddress this,the InternationalSociety ofPsychoneuroen- docrinology(ISPNE)hasinitiated anexpert paneltosummarize relevantevidenceandcollectiveexperienceonmethodologicalfac- torsaffectingCARassessment. Thegoalofthisinitiative wasto formulateclearconsensusguidelinesbasedoncurrentknowledge forfuture studiesinthisevolvingfield ofresearch.Thepresent report summarizes the results of this undertaking. As a large proportionofCARresearchusessalivarycortisolassessmentsin participants’domesticsetting,aparticularfocusisputonmethod- ologicalchallengesinthisresearchcontext.Giventheimportance ofsampletimeaccuracy,thefirstthreesectionsaredevotedtoan in-depthdiscussionofthistopic,includingstrategiestoincrease samplingaccuracybymaximizing participantadherence. Inthe subsequentsectionsarangeoffurthermethodologicalfactorsare covered.Inthefinalsection,thederivedconsensusguidelinesare outlinedandexplained.

2. Cortisolawakeningresponse

TheCARisexpressedaspartofnormal,healthyhumancircadian physiology.DeviationsfromatypicalCARpatternareassumedto markmaladaptiveneuroendocrineprocesses.Ageneralreviewof psychosocial,psychiatricandhealth-relatedcorrelatesoftheCARis beyondthescopeofthisarticle(reviews:ChidaandSteptoe,2009;

Clowet al.,2004;Fries et al.,2009;Kudielka and Wüst,2010).

However,asaprerequisiteforinterpretingsuchdata,somedis- tinctfeaturesoftheCARneedtobeacknowledged.Itisimportant toemphasizethat,althoughhistoricallytheterm‘CAR’hasbeen usedtodescribedifferentaspectsofpost-awakeningcortisolsecre- tion(includingoveralllevels),onlythedynamicofpost-awakening cortisolsecretionisaccuratelyreferredtoasthe‘CAR’,i.e.,corti- solchangesoccurringduetotheawakeningresponse(Clowetal., 2010).Arationaleforthisrecommendedterminologyislaidoutin Section2.2.

2.1. Descriptionanddistinctivefeatures

TheCARrepresentsasharpincreaseincortisollevelsacrossthe first30–45minfollowingmorningawakening.Inhealthyadults, themagnitudeoftheCARwasfoundtorangebetweena50and 156%increaseinsalivarycortisollevels(Clowetal.,2004).Fig.2a depictspost-awakeningcortisolprofilesofchildren,adolescents andelderlyadultsfromthefirstsystematicdescriptionoftheCAR byPruessneretal.(1997).

Theinitialfindingsuggestingthattheawakeningprocessstim- ulatescortisolsecretionprovidedanexplanationforpreviousdata ofpoortest–retestreliabilityinclock-timebasedcortisolassess- mentsduring theearlymorning hours(e.g., Costeet al., 1994;

SchulzandKnabe,1994)andsuggestedthatalignmentofcortisol samplingwith awakeningwould provide a more reliablemea- sure.Thisnotionwassoonsupportedbydatashowingimproved test–reteststabilityofawakening-alignedpost-awakeningcortisol levelsacrossabroadagerange(rsbetween.39and.67;Pruessner et al., 1997).These initialdata were viewed asindicating that theCARcouldbeusedasareliabletraitbiomarkerand,hence, investigationsoverthefollowingyearsmainlyfocusedoninter- individualvariabilityinCARprofilesusingcross-sectionaldesigns.

Such researchrevealedtheCAR toberelated tovarious physi- caland mentalhealth variables,albeitwithsomeinconsistency (review:ChidaandSteptoe,2009).Morerecentevidenceillustrated thattheCARalsoexhibitsconsiderableintra-individualvariabil- ity(see Section7.2).Indeed,although twinstudiesconsistently foundamoderateheritabilityoftheCAR(Kupperetal.,2005;Wüst etal.,2000a),theexpressionoftheCARonaparticulardayhas beenestimatedtobemoreinfluencedbystatefactorsthanbysta-

ble,trait-likeinfluences,includinggeneticfactors(Almeidaetal., 2009;Hellhammeretal.,2007;Stalderetal.,2010b).Buildingon thesedata,researchalsoincreasinglysetouttoinvestigatestate correlatesoftheCAR(review:Lawetal.,2013).

TheCARperiodisembeddedwithinawell-describedcircadian patternofcortisolsecretion,characterizedbyacortisolincrease priortoawakening,theCARperiodandadeclineofmeancorti- sollevelsovertheremainingdiurnalphase(Veldhuisetal.,1989;

Weitzmanetal.,1971).Importantly,thereisconvergingevidence suggesting that the CAR is relatively distinct from earlier and latercomponentsofcircadiancortisolsecretion.Sleeplaboratory researchrevealedthattheCARisnotamerecontinuationofthe pre-awakeningcortisol increase but comprises a superimposed responsetoawakening(Wilhelmetal.,2007;seeFig.2b).Inaddi- tion,theCARwasfoundtobeunrelatedtocortisollevelsduringthe remainderoftheday(Edwardsetal.,2001a;Mainaetal.,2009)or toalatenttraitcortisolfactorinferredfromvariousdiurnalsamples (Doaneetal.,2015).

Evidence showingthat in healthyhumans the CAR, but not laterdiurnalcortisolsecretion,issensitivetolightexposurefur- therillustratesitsdistinctnature:morningawakeningindarkness ordimlight reducesthedynamic of theCARrelative toawak- eninginlight (Figueiroand Rea,2012;Scheerand Buijs,1999).

Furthermore,winterawakeningusingadawnsimulator(gradually increasinglightlevelsbeforeawakening)hasbeenassociatedwith increasedpost-awakeningcortisolproduction(Thornetal.,2004).

Inrodentstudies,light-inducedeffectsonglucocorticoidsecretion wereabsentfollowinglesionsofthesuprachiasmaticnucleus(SCN) orinSCN-intactmicewithadrenalsympatheticdenervation(Buijs etal.,2003).Thus,anSCN-mediatedextra-pituitarypathwayhas beenimplicatedinregulationoftheCAR,butisunlikelytoaffect cortisolsecretionovertheremainderoftheday(review:Clowetal., 2010).

Besidesanuncouplingfrombasalcircadiancortisolsecretion, researchalso consistently revealed the CAR tobe unrelated to cortisolreactivitytoexperimentally-inducedpsychologicalstress (Bouma et al., 2009; Schmidt-Reinwald et al., 1999). This has important implications for the interpretation of CAR data, i.e., indicating itsdistinctness from cortisolreactivity toacute psy- chological stress. Interestingly,in an earlystudy, the CAR was foundtobecloselyrelated tothecortisolrisefollowing ACTH- challenge(r=.63),suggestingthatitsexpressionmaybeinfluenced bythemaximalcapacityoftheadrenalcortextoproducecortisol (Schmidt-Reinwaldetal.,1999).

Overall,thesedatahighlightthedistinctnatureoftheCARand suggestthatitsassessmentprovidesaddedinformationthatmay notbederivedfromothercortisolmeasures.Thistogetherwith findingsofuniqueassociationswithpsychosocial,cognitiveand health-relatedparametersmakestheCARaninterestingmeasure inPNEresearch.Conversely,thesedataalsoillustratethatwhen interpretingrespectivefindings,researchersneedtobecarefulnot tomistaketheCARforeitheramarkerofgeneralHPAaxisactiv- ity/basalcortisolsecretionorstress-reactivecortisolchanges(Clow etal.,2010).

2.2. Maincomponents

TheCARisa dynamicphenomenon triggeredbytheprocess ofmorningawakening.Strategiesforquantifyingpost-awakening cortisolsecretionneedtoaddresstwomainunderlyingcompo- nents:First,thestartingpointoftheCARperiod,i.e.,thefirstsample synchronizedwiththe momentof awakening(S1).Second,the actualdynamicofthecortisolincrease afterawakening,i.e.,the CARitself,assessedatsetintervalsafterawakening.Importantly, thetwocomponents(S1andCAR)areoftenfoundtobeinversely related,withalowerCAR followingahigher S1,and viceversa

Fig.2.(a)Mean(±SEM)post-awakeningcortisolvaluesforthreeagegroupsfromthefirstsystematicdescriptionoftheCAR(fromPruessneretal.,1997).(b)Sleeplaboratory datashowingtheCARtobesuperimposedonthepre-awakeningcortisolincrease(fromWilhelmetal.,2007).Bothfiguresreproducedwithpermission.

(Adametal.,2006;Bäumleretal.,2013;Huberetal.,2006;Stalder etal.,2009,2010b;Wilhelmetal.,2007;Wüstetal.,2000b).This relationshipcanlikelybeinterpretedasanillustrationofthelaw ofinitialvalue(Wilder,1962).

Clowetal.(2010)reviewedneurophysiologicalevidenceonthe regulationofmorningcortisolsecretionandconcludedthatdiffer- entialprocessesarelikelytobeimportantforthepre-awakening cortisolincrease andfor theCAR(Clowetal., 2010).Theythus suggestedthatseparateresultsshouldbereportedforS1(i.e.,the endpointofthepre-awakeningincrease)andestimatesoftheCAR.

Thisisalsorecommendedaspartofthisconsensusreport(seeSec- tion7.4).Inaddition,quantificationstrategiesareinuse, which combineinformationofS1andtheCAR,thusprovidinganindex ofoverallcortisolsecretionoverthepost-awakeningperiod(e.g., theAUCG,Pruessneretal.,2003).Whenusingsuchmeasures,itis importanttoacknowledgethattheyareinfluencedbybothunder- lyingcomponents(S1andtheCAR).Hence,itisimportanttorefer torespectivemeasuresasreflectingtotal‘post-awakeningcortisol concentrations’orsimilar,butnotasmeasuresoftheCAR(Clow etal.,2010).Inlinewiththisreasoning,graphicalillustrationsin thepresentarticlefocusondepictingS1andameasureoftheCAR, inthisinstancetheareaunderthecurvewithrespecttoincrease (AUC_I,Pruessneretal.,2003).Adiscussionofstatisticalapproaches toquantifyingtheCARisprovidedinsection7.4.

3. Inaccuratesampling:prevalenceandimpact

ThevalidityofCARdatacriticallyreliesonthetemporalaccu- racyofsalivasamplingacrossthepost-awakeningperiod.Atypical samplingscheduleinvolvestakingafirstsampleimmediatelyafter awakeningfollowedbyrepeatedassessmentsatspecifiedtimes, e.g.,at10or15minintervalsoverthesubsequent30–60min.Fig.3a illustratesanexemplaryCARsamplingschedule.Failuretocomply withsuchaschedulecanoccurinmultipleways.Inthefollowing, wedistinguishbetweenparticipants(i)failingtocorrectlyreport theirawakeningtimeand/ordelayingtheinitiationofsamplingin relationtothemomentofawakeningand(ii)notadheringtothe specifiedtimeintervalsforlatersampling.

3.1. Delaybetweenawakeningandinitiationofsampling

Thecommencement ofsamplingimmediatelyafter awaken- ingiscrucialforaccuratelycapturingtheCAR.Table1aprovides an overview of studies examining the impact of delayed ini- tiation of samplingafter awakening. Thisresearch employed a rangeofmethods,suchasactigraphy,electrocardiography(ECG) or polysomnography (PSG), to verify participants’ self-reported timesofawakening(seeSection4.2.1foradescriptionofmeth-

ods).Inaddition,tworecentstudiesalsousedelectronicmonitoring devicestoverifytimesofsamplecollection(GriefahnandRobens, 2011;Smythetal.,2013).

Thefirstdescriptionofawakeningtime-relatedsamplinginac- curacywasmadeinapost-hocanalysiscarriedoutonasubgroup of individuals (13.1% of the total sample) who failed to show anyevidence ofa positive CAR (Kupperet al.,2005).By utiliz- ingavailableECGandactigraphydata,itwasrevealedthatthese participantsshowedameandelayof42min(range:10–135min) betweenverifiedandself-reportedawakeningtimes.Bycontrast, participantswithregularCARprofilesmostlyshowedgoodcor- respondencebetweenself-reportedandverifiedawakeningtimes (Kupper et al., 2005). Following these initial data, subsequent researchconfirmedthatfailuretocorrectlyreportthetimeofawak- eningand/ortodelaythebeginningofsamplingafterawakening isrelativelycommonandprofoundlyimpactsCARestimates(see Table1a).Acrossstudies,meanverifiedawakeningtimespreceded meanself-reportedawakeningtimesby3.3–6.2minandmeanself- reportedtimesofcollectingS1by7.1–24.8min(DeSantisetal., 2010;Dockrayetal.,2008;Okunetal.,2010).Aparticularlystriking illustrationofthepotentialextentofsuchinaccuracywasreported byGriefahnandRobens(2011)whoaccumulateddatafromthree studies,eachemployingcarefulobjectiveverificationofawakening andsamplingtimesacross6–8daysperindividual.Theyfoundthat participantsdelayedcollectingS1by3–30minon19.3%ofsampling daysandbyeven>30minon14.0%ofsamplingdays(Griefahnand Robens,2011).

However,delayingthecollectionofS1afterawakeningbymore than15minresultsinfalse-highestimatesofS1andfalse-lowesti- matesoftheCAR.Thispatternemergedbothfromresearchrelying onself-reportsofS1timing(DeSantisetal.,2010;Dockrayetal., 2008;Okunetal.,2010)andfromstudiesobjectivelymonitoring samplingtimes(GriefahnandRobens,2011).Fig.3canddexem- plifytheimpactof20and40minsamplingdelays,respectively,on estimatesofS1andtheCAR(AUC_I).

TheimpactofsmallerdelaysinsamplingS1(<15min)hasbeen moredifficulttocapture.Earlierresearchindicatednodifferences inCARestimatesbetweenfullyaccurateindividuals(delays<1min) andthosewith1–15mindelays(Dockrayetal.,2008;Okunetal., 2010).Otherstudies,however,reportedatrendforanattenuated CAR inindividuals with5–15mindelays(DeSantiset al.,2010) orsuggestedthatCARestimatesalreadystartedtodecreasewith delaysexceeding∼10min(GriefahnandRobens,2011).Animpor- tantadditiontothesedatacomesfromrecentresearchbySmyth etal.(2013,2015):employingcarefulcontrolofawakeningand samplingtimesinhealthyparticipants(samplingat5mininter- vals),theirfindingsrevealedthatminordelays(5–15min)yielded estimatesofanincreasedCARandanearlierpeak.Thishasbeen

Table1 OverviewofresearchexaminingtheprevalenceandimpactofCAR-relatedsamplinginaccuracy. StudySample&designObjectivecontrolmethodSamplinginaccuracydescriptivesImpactonCAR (a)Objectivecontrolofawakeningtime(andsamplingtimes): Kupperetal.,2005N=59,subgroupwithnegative CAR,1dayAW:ECG&actigraphy Sampling:-Objectivevs.self-reportedAW:42mindelay (range:10–135min)NegativeCARacrossgroup Dockrayetal.,2008N=83elderly,suspectedCAD patients,1dayAW:Wristactigraphy Sampling:-Objectivevs.self-reportedAW:6.1±14.8min ObjectiveAWvs.S1:12.2±20.3minGreaterS1&no15–30minincreaseinpps. with>15mindelaybtw.objectiveAWandS1 Okunetal.,2010N=207,elderly, heterogeneous,1dayAW:PSG Sampling:-Objectivevs.self-reportedAW:3.3±53.1min ObjectiveAWvs.S1:24.8±32.2minGreaterS1&smallerCARinpps.with>15min delaybtw.objectiveAWandS1 DeSantisetal.,2010N=91,lateadolescents,3daysAW:Wristactigraphy Sampling:-Objectivevs.self-reportedAW:6.2±14.3min ObjectiveAWvs.S1:7.1±15.9minSmallerCARsinpps.with>15mindelaybtw. objectiveandself-reportedAW Griefahn&Robens,2011N=108(from3studies), heterogeneous,6–8daysAW:PSGoractigraphy Sampling:EMD/verifiedby experimenter(sleeplab) ObjectiveAWvs.objectiveS1:≤3mindelay: 66.7%days,>3and30mindelay:19.3%days, >30mindelay:14.0%days

SmallerCARondayswitha>∼10mindelay btw.objectiveAWandobjectiveS1 (descriptiveanalysis) Smythetal.,2013N=50(studyI),healthy females,4daysAW:Actigraphy Sampling:EMDObjectiveAWvs.objectiveS1:4mindelayGreaterCARondayswith5–15mindelay comparedto<5min (b)Objectivecontrolofsamplingtimes: Kudielkaetal.,2003N=47,community-dwelling subjects,1dayAW:- Sampling:EMDObjectivevs.self-reportedsampling:13±4 min(incl.diurnalsamples)aSmallerCARwithinaccuratesampling(S1: ≥10mindelay,30min:±7mindelay) Brodericketal.,2004N=66,femalefibromyalgia patientsandcontrols,7daysAW:- Sampling:EMDDelayedsamplingon80%days(incl.diurnal samples)aPresenceofCARondayswithaccurate sampling;noCARriseoninaccuratedays Kudielkaetal.,2007bN=83,elderly,3daysAW:- Sampling:EMD60%ofparticipantswithinaccuratesampling (anyCARsamplingoutsidespecified10min timewindow)onatleastoneday SmallerCARinparticipantswithinaccurate samplingon2(outof3)days Smith&Dougherty,2014N=81,preschool-agechildren (mainsubjects)andtheir parents,2days

AW:- Sampling:EMD44.3%samplinginaccuracy(anyCARsample outsidespecified10mintimewindow)of parentssamplingintheirchildren

GreaterS1&smallerCARinchildrenofparents withinaccuratesampling Goldenetal.,2014N=935,multi-ethnicsample, mixedgender,3daysAW:- Sampling:EMDSamplingdelay>15minin21.0%ofS1samples and31.2%of30minsamplesSmallerCARinparticipantswhoscoredinthe lowesttertileofoverallcompliance Abbreviations:AW,awakeningtime;btw,between;CAR,cortisolawakeningresponse;ECG,electroencephalography;EMD,electronicmonitoringdevice;pps.,participants;PSG,polysomnography;S1,firstsampleonawakening. aFurtherqualificationoffactorsdeterminingadherencerateswasprovided(seedetaileddiscussioninthetext).

Fig.3.IllustrationofanexemplaryCARprofileandtheimpactofdelayedbeginning ofsamplingafterawakeningontwocommonmeasuresofpost-awakeningcortisol secretion,i.e.,thefirstsampleonawakening(S1)andtheareaunderthecurvewith respecttoincrease(AUCI,Pruessneretal.,2003).Thefigureshowsestimatesgiven (a)correctsamplingandfordelaysof(b)8min,(c)20minand(d)40minbetween awakeningandcollectingS1.Latersamples(S2–S4)areshowntobecollectedinline withthespecified15minintervals.

accountedforbytheobservationthatcortisollevelsremainedrel- ativelyunchangedoverthefirst5–10minpost-awakening(‘latent period’),witha significantincrease firstbeingdetectableinthe 15minsample.Thesedatasuggestthatmoderatesamplingdelays shift the examined time window closer to the actual increase componentbyremovingthelatentperiodfromtheanalysis,thus resultinginhigherCARestimateswithanearlierpeakcomponent (Smythetal.,2013,2015).Fig.3billustratesthisnotionforanexem- plary8minsamplingdelay.

Inaccuracy in the commencement of sampling immediately after awakening can arise from a range of scenarios, includ- ing non-adherence due to motivational reasons (avoidance of discomfort, attending to other responsibilities, etc.). However, observationsbyGriefahnandRobens(2011)aswellasbySmyth etal.(2013)suggestedthatnon-motivationalfactorsmightalso

influenceawakening-relatedinaccuracies.Inbothstudies,consid- erablesamplingdelaysoccurredforS1eventhoughparticipants tooklatersamplesincloseaccordancewiththeprotocol.Thissug- geststhatdelayedsamplingafterawakeningmaybetheprimary causeofinaccurateCARassessmentandariseinwell-intentioned andotherwiseconscientiousparticipants(Smythetal.,2013).A potentialexplanationforthisistheoccurrenceofsleepinertiain theimmediatepost-awakeningperiod,i.e.,astateofreducedcogni- tiveandmotorperformance(TassiandMuzet,2000).Sleepinertia mayincreasethedifficultyofadheringtorequestedtimingsand/or mayimpedetheprecisedeterminationofthemomentwhenoneis fullyawake(Clowetal.,2010;Smythetal.,2013).

In sum,recent evidencesuggeststhat even well-intentioned participantsmay not alwaysbe able toprecisely identify their awakeningmoment.Thiscanleadtomoderatedelaysincollect- ingS1afterawakeningthataresufficienttosubstantiallybiasCAR estimates.Inlightofthis,objectiveverificationofawakeningtime isnecessaryforobtainingvalidCARdata.

3.2. Inaccuratepost-awakeningsampling

BesidesfailuretocollectS1immediatelyonawakening,inac- curacymayalsoarisefromdelaysatsubsequentsamplingtimes.

Table1bsummarizesdataonthecorrespondencebetweenself- reported and objectively verified times of saliva sampling in ambulatoryCARresearch.Twolandmarkstudiesfocusedonsam- pling accuracy during the post-awakening and the remaining diurnal samplingperiod(Broderick etal., 2004;Kudielka etal., 2003).Inaccuratesalivasamplingoccurredrelativelyfrequently andwasassociatedwithanunderestimationoftheCAR.Thisgen- eralpatternwaslaterconfirmedbyresearchspecificallyfocusing ontheCARinadults(Kudielkaetal.,2007b),inparentsobtaining CARsamplesoftheirpreschool-agechildren(SmithandDougherty, 2014)and ina largemulti-ethnicsample (Goldenet al.,2014).

Animportant qualitative extension of thesedata wasprovided byfindingsshowingthattheaccuracyofsalivasamplingcanbe considerablyimproved byinformingparticipantsaboutthefact thattheyarebeingobjectivelymonitored(Brodericketal.,2004;

Kudielkaetal.,2003).Thepotentialimplicationsofthislatterfind- ingarediscussedindetailinSection4.2.4.

4. Strategiesfordealingwithinaccuratesampling

Thefollowingsectionsdescribeavailableobjectivemonitoring strategiesforambulatoryCARresearch,discusswaysfordealing withidentifiedinaccuratedataandlookintopotentialstrategiesin lieuofobjectivemeasures.

4.1. Objectivemonitoringstrategies

AccurateassessmentoftheCARrequiresobjectiveverification ofawakeningandsamplingtimes(AdamandKumari,2009).Ide- ally,suchobjectivemethodsshouldbeemployedincombination withadiarylogsystemtorecordself-reportdataofawakeningand samplingtimes(besidesotherfactors,suchaspotentialcovariates;

seeSection6.3).

4.1.1. Methodsforverifyingawakeningandsamplingtimes

Severalmethods havebeenused toverifyawakeningtimes.

Polysomnography(PSG)isconsideredthegoldstandardinsleep research(VandeWateretal.,2011)andhasbeenusedforveri- fyingawakeningtimesinCARresearch(e.g.,Gribbinetal.,2012;

Griefahnand Robens, 2010,2011; Okunet al.,2010).However, PSGiscostly,labor-intensiveanddisruptivetoparticipants’nor- malroutines(VandeWateretal.,2011).Wristactigraphymight beamorereadilyobtainablemethodasitisminimally-disruptive,

relativelyinexpensiveandwell-validatedagainstPSGforassess- ingsleepparameters(e.g.,Coleetal.,1992;Lichsteinetal.,2006).

Wristactigraphyhasbeensuccessfully usedinambulatory CAR researchacrossseveralstudies(e.g.,Smythetal.,2013).Another actigraphy-basedapproachistheuseofchest-wornmotilitymon- itorsthatadditionallyrecordheart inter-beat-interval(IBI)data (CARresearch:Kupperetal.,2005;Stalderetal.,2011).Asarousing fromsleepisassociatedwithanincreaseinheartrate(e.g.,Huikuri etal.,1994)andrapidcardiovascularactivationlastingaroundten heartbeats(Trinderetal.,2001,2003),availableIBIdata(together withactigraphydata)couldfurtherhelptomorepreciselydeter- minetheawakeningmoment.Still,eachoftheabovedescribed methodsappearssuitablefortheverificationofawakeningtimein CARresearch.Inaddition,futureresearchmayexploretheuseof recentlydevelopedsmartphone-linkedorconsumer-branddevices aspotentiallowcostalternativesforobjectiveawakeningtimever- ification(review:Kellyetal.,2012).However,thisstronglyrestson thesuccessfulvalidationofsuchdevicesagainstawell-established method,suchasPSG,whichtodateismostlystilllacking(Kelly etal.,2012;Meltzeretal.,2015).

Concerningtheverification ofsamplingtimesin ambulatory research,thecommonlyusedelectronicmonitoringsystemshave provenuseful.Thesetypicallyusescrewtopbottles thatrecord timesofbottleopenings,however,boxesthatrecordtimestamps havealsobeendevised.Bystoringsalivasamplingdevicesinside thebottleandinstructingparticipantstorestrictopeningstothe timesofsampletaking,therespectivetimestampsprovideanindi- rectindexofsamplingtimes.Clearly,theuseofsuchsystemscannot fullyprotectagainstintentionalmisuse(e.g.,participantsmaystill takeoutsamplesfromthebottlewithoutperformingthesaliva sampling)butitdoespresentthecurrentbestpractice.Alternatives tothisapproachmightariseasaconsequenceofmoderntechnol- ogy.Forexample,smartphoneswithbuilt-incamerascouldbeused toobtaintime-stampedself-photographs(‘selfies’)byparticipants whencollectinga sampletoverify samplingaccuracyin future studies.Ifadequatelydeveloped,suchastrategycanequallyberec- ommendedfortheverificationofsamplingaccuracyaselectronic monitoringsystems.

Analternativeapproachthatremovestheneedforsampling time verification isthe useof automatedsamplingmethods to assesstheCAR.Forone,intravenousbloodsampling,whencou- pledwithstationaryPSGassessment(e.g.,Wilhelmetal.,2007), ensurestheaccuracyofCARassessment.However,asthisresearch istypicallyrestrictedtothesleeplaboratory,itsartificialsettingis associatedwithreducedecologicalvalidity.Thismaybeprevented byrecentlydevelopedsystemsforautomatedsamplingofsubcuta- neoustissuefreecortisol(Bhakeetal.,2013).Althoughclearlymore demandingandinvasiveforparticipantsthantheself-collectionof salivasamples,thisapproachcouldpotentiallyallowtheassess- ment ofthe CAR in participants’ homesettingsin some future research.

4.1.2. Dealingwithverifiedinaccuratedata

Onceinformationonsamplingaccuracyhasbeenobtained,it canbeusedtoreducebiasonCARestimatesthrough(i)dataexclu- sionstrategiesand(ii)statisticalmodelingapproaches.

Fordataexclusionstrategies,theextentofinaccuracyisusu- allyfirstcalculatedasthediscrepancybetweenthescheduledand theactual/verifiedsamplingtime(t).Thet-valueofindivid- ualsamplingtimesisthencomparedtoapredeterminedaccuracy margin(e.g.,5,10or15min)and,incaseanytexceedsthismar- gin,CARdatafortherespectivesamplingdayareexcludedfrom subsequentanalyses(e.g.,Kudielkaetal.,2003).Whenusingsuch anapproach,decidingonthemostsuitableaccuracymarginfor dataexclusion is difficult.Proceedingfromtheabove reviewed findings(particularly:Smythetal.,2013,2015),evensmalltime

discrepanciesmayentailsubstantialbiasonCARestimates,unless theybecomenegligible(i.e.,t≈0min).However,narrowingaccu- racy margins causes a growing loss of (putativelyinformative) data.Consequently,anyconsensusaboutafixedaccuracymargin isnecessarilyatrade-offbetweenscientificprecisionandpractical feasibility.Forexample,previousresearchemployingawakening timeverificationbywristactigraphysuggeststhatspecifyingan accuracy margin oft=0±5minfor S1 will yielddata loss of 26–46%(DeSantisetal.,2010;Dockrayetal.,2008).Inaddition, theselectiveexclusionofparticipantswithinaccuratesampling mayresultinpotentialselectionbiasandreducedgeneralizability ofresults.Inordertokeepthepercentageofclassifiedinaccurate data(andthusdatalossandpotentialbias)aslowaspossible,itis crucialtoemployafullrangeofmeasurestomaximizeadherence (seeSection5).

Inthesecondgroupofstrategies,verifiedinaccuratedataare notexcludedbutinsteadtheobjectiveinformationonactualsam- plingtimesisutilizedforthecalculationofCARestimates(i.e.,these dataareincorporatedintothestatisticalmodel).Hence,thispro- videsamoreeconomicalapproach,preventingunwanteddataand participantloss,andresultingconcernsregardingreducedgener- alizability.Tousesuchastrategy,statisticalmodelsarerequired thatadequatelydescribethetemporaldynamicsofcortisolsecre- tionacrosstheCARperiod.Section7.4providesadescriptionof suchmodellingapproachesinCARresearch.

Insum,thedecisionaboutthemostadequatestrategyinvolves tradingoffconsiderationsaboutscientificprecisionagainstthose ofpracticalfeasibility.Researchers’primaryconcernshouldbeto obtainvalid,unbiaseddata.Theuseofawell-specifiedstatistical modeloftheCARthatincorporatesverifiedawakeningandsam- pling time datafulfils this criterion and should bethemethod of choice. Whenusing data exclusion strategies, achieving any confidence thatCAR dataare notbiased requiresthe specifica- tionofrelativelystrictaccuracymarginswhich,unfortunately,is associatedwithdataloss.Toachievecomparabilitybetweenstud- ies,werecommend that futureresearchemploysa transparent approach whereby it is clearlystated whetherfindings emerge whenapplyingastrictaccuracymarginoft=0±5minforeach post-awakeningsample,eitherasthesoleapproachorincombina- tionwithresearchers’ownanalyticalstrategy(i.e.,asanadditional sensitivityanalysis).

4.2. Arethereviablestrategieswithouttheuseofobjective measures?

Theuseofobjectivemonitoringstrategiesincreasesthecosts perparticipantandmaythusreducethenumberofparticipants fromwhomendocrinedatacanbeobtained(AdamandKumari, 2009).Thismayleadresearcherstoconsiderwhetheralternative strategiesexistthatyieldvalidCARdatawithouthavingtoemploy objectivemeasures.

4.2.1. Forcedawakening

Adesign-based strategytocounteract problems ofsampling inaccuracyistoexternallyawakenparticipants(usuallythrough study personnel). This is a work-intensive approach that has beenusedwithparticipantsexaminedinahospitalsetting(e.g., Huberetal.,2006;NicolsonandVanDiest,2000),sleeplabora- tory(Wilhelmetal.,2007)orquarantinedaspartofalargerstudy (Polketal.,2005).Recently,avariationofthisapproachhasbeen employedininfantsandyoungchildrenwhoweretooyoungto samplesalivathemselvesandwerethuswokenupbytheirparents toensuretheaccuracyofsamplinginitiation(Bäumleretal.,2013;

Bäumleretal.2014a;Bäumleretal.2014b;Stalderetal.,2013).

Inthelatterstudies,thiswasfurthercomplementedbyobjective verificationofawakeningandsamplingtimes.

Anargumentinfavorofaforcedawakeningapproachisthat currentevidencesuggeststhattheCARis unaffectedbypartici- pants’modeofawakening(spontaneousvs.externallywoken;e.g., byalarmclock;Stalderetal.,2009;Wüstetal.,2000b).Thismakes itunlikelythatforcedawakeningleadstofundamentallydifferent CARprofilesthanspontaneousawakening.However,aremaining dangeristhatparticipantsmaywakeuppriortotheplannedwake uptime.Hence,aforcedawakeningapproachshouldstillbecom- plementedbyobjectiveawakeningtimeverification.Undersuch acondition,forcedawakeningmayhelptoyieldhighqualityCAR data,particularlyifstudypersonnelalsocontinuetomonitorthe accuracyofsubsequentsalivasampling.Thelatterpossibilitymay thensparetheuseofelectronicmonitoringdevicestoverifysam- plingaccuracy(seeSection4.2.1).

Besidesissuesrelatedtosampletiminginaccuracy,however, researchersassessingtheCARinahospitalorsleep-laboratoryset- tingneedtobeawareofthepossibilityofstate-relatedconfounding whichcaninducesignificantbiasinCARanalyses(seeSection6.2).

Further,asmentionedbefore,suchconditionsareassociatedwith reducedecologicalvalidity,which isa keyadvantageofsample collectioninparticipants’homesettings.

4.2.2. Increasingstatisticalpower

Researchersmaywonderwhetherproblemsofsamplinginac- curacycannotbeovercomebysimplyincreasingstatisticalpower, e.g., in large-scaleepidemiological research.Indeed, this would bethecaseifinaccuracyoccurredrandomly,i.e.,notsystemati- callyrelatedtorelevantparticipantcharacteristics(cross-sectional research)orsituationalfactors(intra-individualresearch).Under suchcircumstances,inaccuratesamplingwould merelyincrease theerror ofCAR estimates, whichcould betackledbyincreas- ingthenumberofobservations.However,extensivedataindicate that non-adherence, a factor which is likely to strongly affect samplinginaccuracy,doesnotoccurrandomlybutcovarieswith relevantpsychologicalfactors.Forexample,researchhasshown thatadherencetomedicaltreatmentsregimensisinfluencedby individual differences in depressiveness and/or social support (meta-analyses:DiMatteo,2004;DiMatteoetal.,2000).Research focusingontheCARalsoconfirmedaninverserelationshipbetween perceivedsocialsupportandsamplinginaccuracy(Kudielkaetal., 2007b).In a largemulti-ethnic study,inaccurate samplingof a diurnal cortisolprofile(including post-awakeningsamples) was relatedtolowerincome,educationlevels,and(marginally)eth- nicity (Golden et al., 2014). Furthermore, effects of sampling accuracyhavebeenfoundtointeractwithhealthstatus,i.e.,female fibromyalgiapatientswerelessinfluencedbybeinginformedabout theuseofobjectivemonitoringstrategiesthanhealthy controls (Brodericketal.,2004).Thisindicatesthatinaccuratesamplingis likelytoco-varywithparametersthatareofcentralinteresttoPNE inquiry,i.e., psychologicalorhealth-related factors.Under such circumstances,failingtocontrolforsamplingaccuracyposesthe eminentthreatthattruerelationships maybeobscuredorfalse relationshipsmaybeaccepted.Inthiscase,“(i)ncreasingtheNor thenumberofsamplescollectedwillyieldthesamelevelanddirection oferror.Infact,increasingstatisticalpowerwouldonlyincreasethe researcher’sconfidenceinafalseresult.”(Brodericketal.,2004;p.

648).

Thereislessevidenceonstatecorrelatesofsamplinginaccu- racy fromintra-individualCAR research.Brodericket al.(2004) observednodifferencesinsamplingaccuracybetweenweekdays and weekends, a factor frequentlyassociated with altered CAR profiles(Kunz-Ebrechtetal.,2004b;Schlotzetal.,2004).Still,it isclearlyconceivablethatinaccuratesamplingmayco-varywith statepsychologicalfactorsrelevant for PNEresearch(e.g.,state arousalorstress,prospectivememoryload,sleepcharacteristics;

Lawetal.,2013).Hence,itcannotbeexcludedthatfailuretoobjec-

tivelycontrolforinaccuratesamplingconfoundsintra-individual CARdata.Again,thisproblemcannotbealleviatedbyincreasing statisticalpower.

4.2.3. ExclusionofCARnon-responders

Inaccuratesamplinghasoftenbeenassociatedwithflattened orevennegativeCARprofiles(e.g.,Brodericketal.,2004;Dockray etal.,2008;Kupperetal.,2005).Thishasledtotheproposition thatissuesofsamplinginaccuracymaybeaddressedbyexclud- ingparticipantswhofailtoshowa cortisolincreasefromS1to latersamplesastheseare‘suspectednon-adherents’(Thornetal., 2006).However,thisisunlikelytobeasufficientapproach.The effectsofinaccuratesamplingonCARestimatesarelikelytobenon- linearandcontinuous(seeFig.3b–d):comparedtofullyaccurate sampling,smalldelaysafterawakeningmayfirstresultinanover- estimationoftheCARwhichthenturnsintothewell-documented underestimationoftheCARwithlongerdelays(>15–20min).The complete absenceofa post-awakeningincrease (i.e.,anegative CAR)islikelytooccuronlyifthedelaybetweenawakeningand theinitiationofsamplingexceedsthepeakoftheunderlyingCAR (between30 and45min,seeFig.3d).Theexclusionofnegative CARprofileswouldthuseliminateonlyextremecasesofinaccu- ratesamplingbutnotmildormoderatecases,whichalreadyhave thepotentialtosubstantiallybiasresults.

Inaddition,theexclusionofCARnon-respondersmaybyitself inducebias.Itisstillunresolvedwhether,givenfullyaccuratesam- pling,therearegenuineoccurrencesofparticipantsnotshowinga positiveCAR.Preliminaryevidencesuggeststhatthismayindeed bethecase:inaccuratelysampleddatabasedonobjectivemoni- toring,noincreaseoronlyaminorCAR(<2.5nmol/L,Wüstetal., 2000b;or<1.5nmol/L;Milleretal.,2013a)emergedon13.1%of studydays ininfants(Stalder etal., 2013), on18.0% ofdays in toddlersandyoungchildren(Bäumleretal.,2013), inadultson 14.7%(Dockrayetal.,2008),andon19.7%ofdaysinhealthypar- ticipants(Smythetal.,2013).Ofnote,patientswithbrainlesions, particularlyinthehippocampalformation(Buchananetal.,2004;

Wolfetal.,2005),appeartoshowmoregenerallyattenuatedor evenabsentCARs(seealsoSection6.3).Overall,thesedataindicate thatabsentCARsmayrepresentgenuinephenomenathatsimply formthelowerendofadistributionofCARmagnitudes.Following thisassumption,absentCARsshouldoccurmorefrequentlywithin groupsthatasawholeexhibitareducedCARprofile.CARresearch ofteninvestigatesgroupdifferences(e.g.,betweenclinicalpatients andcontrolsubjects),thusbydefinitiontryingtoprovethatone grouphasalowerCARthananother.Hence,aCARnon-responder exclusionstrategywouldbiasdatabysystematicallyexcludinga greaterpercentageoflowvaluesfromonegroupthanfromanother.

4.2.4. Informationalstrategies

Inresearchexaminingdiurnalcortisollevels(includingpost- awakeningsampling),participantswhowereinformedabouttheir samplingaccuracybeingverifiedbyelectronicmonitoringsystems showeda76%reductionincumulatedsamplingdeviationscom- paredto‘non-informed’participants(Kudielka etal.,2003)and moredayswithaccuratesampling(informed:90%,non-informed:

71%;Brodericketal.,2004).Importantly,besidesimprovingthe actual rates of sampling accuracy, ‘informed’ participants also tendedtocorrectlyself-reporttheirsamplingtimes,evenifthey hadnotsampledaccurately(Brodericketal.,2004;Kudielkaetal., 2003).Subsequentresearchwithparticipantsbeinginformedabout objective monitoring mostly confirmed high accuracy of post- awakeningsamplingintheseindividuals (Griefahnand Robens, 2011;Smythetal.,2013)althoughthiswasnotthecaseinarecent studyonalargemulti-ethnicsample(seeTable1b;Goldenetal., 2014).

Table2

Strategiesformaximizingparticipantadherencewiththesamplingschedule^a. Face-to-facestrategies&materials

Increasingparticipantmotivation:

- Engageparticipantswiththeresearchgoalsandclearlyexplaintheimportanceofstrictadherencetothem - SpecificallyemphasizetheimportanceofcollectingS1immediatelyuponawakening

- Informparticipantsaboutthefactthattheirsamplingaccuracyisobjectivelymonitored - Establishappropriatesamplingdatesthatareconsideredas‘convenientandtypical’

- Allowparticipantstoaskquestions

Increasingtheclarityoftheprocedure:

- Gothroughtheprotocolwithparticipantsindetail - Ifpossible,practicerelevantpartswithparticipants

- Provideadditionaltake-homeinstructions(writtenformorasinstructionalvideo/DVD) - Adviseplacingsamplekit(withapen)besidebedthenightbeforesamplecollection - Defineandexplainwhatismeantbythe‘themomentofawakening’

- Makecollectionkituserfriendlyandpopulationappropriate(e.g.,largeprintinelderly) - Helpparticipantstoorganizethecollection(e.g.,usecolorcodedsamplingdevices)

Additionalstrategies

- Calloremailtheeveningbeforesamplingtohighlightinstructionsandremindaboutstartingthenextmorning - Useofautomatedremindertoolsthatareactivatedbyparticipantsonawakening

- Reminderphonecallsortextmessagingatthesamplingtimesthroughresearchpersonnel(onlyappropriateforpopulationswithwell-definedawakening times)

aStrategieswerepartlyadoptedfromAdamandKumari(2009).

Still,theabovedatamayindicatethepossibilitythatmerely informingparticipantsabouttheuseofobjectivecontrolmethods couldprovideastrategyforobtainingreliabledataonsampling accuracy(i.e.,throughself-reports).Inthiscontext,‘mock’strate- giescouldbeconsidered,withparticipantsbeingtoldthatobjective monitoringstrategiesarebeingusedwithoutthisactuallybeing thecase. Besidesethicalconsiderations, it isimportant tonote that theefficacy of such an approach hasnot been tested yet.

Thisisnottrivial astheeffectivenessofa mockcompared toa real‘informed’strategymaybereducedbyseveralroutes,suchas non-verbaltransmissionoflowerexpectationsfromexperimenters toparticipants (givenexperimenters’ awareness that noobjec- tivemonitoringisbeingused)orpassingonofinformationabout thenon-functionalityofobjectivemonitoringbetweenparticipants (e.g.,instudentpopulations).Apotentialsolutionmaybean‘open’

strategyaspartofwhichobjectivemonitoringisemployedinaran- domsubgroup,whileallparticipantsaretoldthatthereisachance ofbeingmonitored(AdamandKumari,2009).Whilethisavoids deceivingparticipants,itisunclearwhetherinformationaboutthe merechanceofbeingmonitoredisequallyeffectiveascertainty aboutthisfact.Hence,withoutfirmevidenceshowingtheeffec- tivenessofsuch anapproach,it isrecommendedthat objective monitoringisemployedacrossallparticipantsandisnotsubsti- tutedwithaninformationalstrategy.Notwithstanding,evidence clearlysuggeststhatparticipantsshouldalwaysbeinformedabout theuseofobjectivemonitoringstrategies.

5. Maximizingadherence

Irrespectiveof objectivemonitoring, it is expedient towork towards maximizing participant adherence. Such strategies are cost-efficientastheypreventdatalossthroughtheexclusionof inaccurately sampled data and increase data quality (i.e., fully adherentdataaresuperiortostatisticallyinferred/correcteddata).

Table2listsstrategiesformaximizingadherenceinCARresearch.

Severalof thesestrategiesarederived fromtheauthors’collec- tiveresearchexperience,withoutformalpublishedevidenceon effectivenesstesting.

Animportantopportunityforincreasingparticipantadherence isprovidedbytheinitialface-to-facemeeting.Strategiesemployed duringthis meetingmaybothraise participants’motivation for beingadherentandhelptoincreasetheclarityofthestudypro- cedure.Animportantwaytoraisemotivationistryingtoengage participantswiththeresearchgoals.Besidesconveyingthegen- eralpurposeofthestudy,thisinvolvesexplainingtheimportance ofbeingadherentinCARresearchandtheconsequencesofnon- adherence.Toensurethatparticipantsfullyunderstandthewhole studyprocedure, itis consideredimportantthat researchersgo throughthe protocolin detail withthem andpractice relevant components(e.g.,thesalivasamplingprocedure).Aspartofthis, itshouldbeexplained preciselywhatismeantbythe‘moment ofawakening’ inorder tostandardize thiscritical aspectacross participants(Adam&Kumari,2009).Werecommendthatsucha definitionshouldfocusontheregainingofconsciousnessasthe centralcharacteristicoftheawakeningmoment(e.g.,“Whenyou areawake, i.e.,you areconscious: youknow whoandwhere you are;youareinastatethatisclearlydifferentfromwhenyouwere sleepingeventhoughyoumaystillfeeltired.”).Inaddition,itshould bemadeclearthatparticipantsshouldnotinitiatesamplingafter prematurenightlyawakenings(e.g.,“Ifyouwakeduringthemid- dleofthenightandplantogobacktosleep,donotbeginsampling;

pleaseonlybeginwhenyouareawakeforthefinaltimebeforeyou plan to getup for the day.”)and that theyshould refrainfrom dozingorsnoozing duringtheCAR samplingperiod(e.g.,“Dur- ingthisstudy,please donot fallback tosleepor‘doze’afteryour initialawakening.Youcanstayinbedorgetoutofbedbutplease stayawake(evenifyouarenotfullyalert)duringandafterthesaliva samplingperiod.”).Besidesusingtheinitialface-to-facemeeting toclarifysuch criticalquestions aboutsample timing,it isalso importantthatappropriatesamplingdatesarenegotiatedbythe researcher and participantand agreed as‘convenientand typi- cal’.

Besidesface-to-facecontact,take-homeinstructionsinwritten formshouldbeprovided.Forsomepopulations,theadditionaluse ofinstructionalDVDshasprovenuseful(e.g.,Stalderetal.,2013).

Overall,itisimportanttomakeinstructionsasexplicitandpractice- orientatedaspossible,e.g.,participantsmaybetoldtoplacethe

samplekitandapenbesidethebedbeforegoingtosleeptoavoid post-awakeningdelaysthroughhavingtosearchforthematerial (AdamandKumari,2009).Strategiesthatmakethecollectionkit moreuserfriendlyandhelpparticipantsorganizethecollection (e.g.,colorcodingofmaterial)arealsodeemedhelpful.Researchers havefurtherhadpositiveexperienceswithusingreminderphone calls,emails,ortextmessagesontheeveningpriortosampling (e.g.,Smythetal.,2013).Besidesremindingparticipantsofimpor- tant procedures (e.g., towear actigraphydevices tobed), such measuresalsosignalanextraeffortmadebytheresearchteam, thusagainhighlightingtheimportanceofaccuratelyfollowingthe studyproceduretoparticipants.Inaddition,recentstudieshave employedmethodsforremindingparticipantsabouttimesofpost- awakeningsamplecollection.Theseincludeautomatedstrategies, e.g.,reminderwatches(Franzetal.,2013),remindingthroughpar- ticipants’mobilephones(Garcia-Bandaetal.,2014)orelectronic reminders,e.g.,timersthatareactivatedbyparticipantswhentak- ingS1andthenbeep/flashatthelatersamplingtimes(e.g.,Doane andAdam,2010;GriefahnandRobens,2011).Inaddition,reminder phonecallsortextmessagingatparticipants’individuallypredicted samplingtimeshasbeenemployed(e.g.,Oskisetal.,2009).While suchstrategiesmayincreaseadherencebypreventingagainstthe forgettingof sampling,theycannot providecertaintyaboutthe accuracyofsampling.Hence,theyshouldonlybeviewedascom- plementaryapproachesbutcannotreplaceobjectivemonitoring strategies.

6. Dealingwithcovariates

Researchershavetodealwiththefactthathormonesecretionis relatedtoalargenumberofstateandtraitfactors(Schlotz,2011).

Dependingontheresearchcontext,thesecovaryingfactorsmaybe consideredconfounders,mediators,moderatorsordirectvariables ofinterest(AdamandKumari,2009;Kudielkaetal.,2012;Schlotz, 2011).Ifacovariateisnotofmaininterest,themostcriticalques- tioniswhetheritconfoundsobservedassociations(Schlotz,2011).

ConfoundingisgivenwhenacovariateisrelatedtoboththeCAR andthevariable(s)ofinterest,thuscreatingaspuriousrelationship betweenthem,andneedstobeaddressed.However,evenifafac- torisonlyrelatedtotheCARbutnottoothervariablesofinterest, thismayincreasetheerrorvarianceofthemodelandthusreduce statisticalpowerfordetectingassociationswiththeCAR(Schlotz, 2011).Strategiesforpreventingunwantedinfluencesofcovariates inambulatoryPNEresearchcanbegroupedintoinstructional,sta- tisticaladjustmentandexclusionstrategies(Kudielkaetal.,2012;

Schlotz,2011).

6.1. Instructionsaboutpost-awakeningbehavior

Besidesinformingparticipantsaboutthenecessity tocollect samplesincloseaccordancewiththespecifiedsamplingtimes(Sec- tions3–5),furtherinstructionsmayaddressparticipantbehavior overthepost-awakeningperiod.Table3providesanoverviewof factorstobeconsideredinthiscontext.Themostcommoninstruc- tionshavebeenforparticipantstotakenilbymouthotherthan water,refrainfromsmokingandomitcleaningtheirteeth(toavoid abrasionandvascularleakageintosaliva)untilafterthefinalsam- pling(Clowetal.,2004).Thereissupportforaninfluenceofthefirst twofactors:Cortisolsecretionisknowntobeacutelyinfluenced bycaffeineandnicotineintake(review:Kudielkaetal.,2009),food consumption(particularlyhighproteinfoods;e.g.,Gibsonetal., 1999;Rosmondetal.,2000)andbloodglucoselevels(Rohleder andKirschbaum,2007).Thissuggeststhatbreakfasting(incl.caf- feinatedorsugareddrinksand/orprotein-richfoods,e.g.,eggs)or smoking duringthepost-awakening phase mayaffect theCAR.

Table3

Guidelinesforinstructingparticipantsontheirbehavioroverthemorningperiod.

Factor: Recommendation:

Specificmodeofawakening(e.g.,onlybyalarmclock) Norestriction Untilhavingfinishedthemorningsamplingroutine:

Drinkingandeating:

- Caffeinateddrinks Disallow^a

- Sugareddrinks Disallow^a

- Food/breakfast Disallow^a

Smoking Disallow^a

Toothbrushing/dentalhygiene Norestriction

Physicalactivity:

- Remainsupine Norestriction

- Restrictmovement Norestriction

- Physicalexercise Disallow^a

Important:participantsshouldalwaysbeencouragedtoreportonengagingin anyofthesebehaviors.

aDependingontheresearchcontext,itmayalsobejustifiabletoallowengage- mentinthesebehaviorsandtostatisticallyadjustforthisaccordingly.

By contrast,toothbrushingis atleastunlikely tobeassociated withstronggroupeffectsassalivarycortisollevelswerefoundto beunaffectedbynormaldentalhygiene(Gröschletal.,2001)or evenvigoroustoothbrushing,despitethelatterleadingtoblood leakageintosaliva(Kivlighanetal.,2004).Researchinchildren, usingsalivarytransferrinlevelsasamarkerofbloodcontamination, alsoconcurredwiththegeneralnotionthatbloodcontamination throughdentalhygieneisunlikelytohaveastrongeffectonsalivary cortisollevels(Grangeretal.,2007).

Similarly,currentfindingsspeakagainstaninfluenceofphysi- calbehavior/activitylevelsinthenormalrangeontheCAR,which hasbeenfoundtobeunaffectedbyposturalchanges(i.e.,remain- ing supine vs.standing/behaving normally; Hucklebridgeet al., 2002;Wilhelmetal.,2007)orthelevelofmotilityoverthepost- awakeningperiod(Stalderet al.,2009).However,thisdoesnot applytophysical exercising,which isknowntoinducecortisol reactivitywhenperformedaboveacertainintensitylevel(e.g.,Hill etal.,2008;KirschbaumandHellhammer,1994).Finally,partici- pants’modeofawakening(spontaneousvs.alarmclock)hasbeen foundunrelatedtoexpressionoftheCAR(Stalderetal.,2009;Wüst etal.,2000b).Still,astheabovestudiesdidnotcontrolforthebeta error,itcannotbeexcludedthatsmalleffectsexistbutwerenot detectedintherespectivestudysamples.

Together, an effect onthe CAR is particularly suggested for eating,drinking(caffeinated orsugared beverages), smokingor engaginginphysicalexerciseduringthepost-awakeningperiod.

Concerningthesebehaviors,inmostresearchcontextsitisrec- ommended that researchers (i) instruct participants to abstain fromthesebehaviorsuntilaftertheyhavefinishedpost-awakening sampling. Alternatively, (ii) in case researchers feel that these restrictionsimposeatoosevereburdenonparticipants’ normal routines(i.e.,reducingwillingnesstoparticipateand/orecological validity),participantsmaybeallowedtoengageinthesebehaviors butshouldthenbestronglyencouragedtoreportthissystemati- cally(e.g.,throughthediarylogsystem).Thisiscriticaltofacilitate subsequent statisticaladjustment for such potential influences.

Thelatterpointalsoappliestothosebehaviorswithoutaproven influenceontheCAR(modeofawakening,dentalhygiene,moder- atephysicalactivity),forwhichitisstillrecommendedtoobtain self-reportdata.Furthermore,ininstanceswhenparticipantsare allowed toeat, drinkand/or brush their teeth,they shouldbe instructedtorinse theirmouthafterwards andtoabstainfrom engagingin thesebehaviorsin theimmediateperiod (1–2min) beforesampling.