Effects of Religious Fasting on Markers of Oxidative Status in Vitamin D Deficient and Overweight Orthodox Nuns versus Implementation of Time-Restricted Eating in Lay Women from Central and Northern Greece

Abstract

Mediterranean diet has been widely suggested to exert significant beneficial effects on endothelial oxida-tive status and cardiometabolic health. Greek Orthodox monasteries, due to their specific nutritionaland sartorial habits, comprise a population which strictly adheres to nutritional patterns with restricted eating and a plant-based subset of the Mediter-ranean diet, often accompanied by profound hypovitaminosis D. Time –restricted eating is also adopted from a large part of general lay Greek population for health promoting reasons, without restrictions on an-imal product consumption, as imposed by Orthodox religious fasting. However, comparative effects of these nutritional patterns on oxidative stress markers remain scarce. The present study attempted to evaluate the effects of Christian Orthodox fasting (COF) in a group of vitamin D –deficient and overweight Orthodox nuns from Central and Northern Greece, compared to the implementation of TRE, 16:8 dietary regimen in a cohort of adult women from the general population from the same region, with regard to markers of endothelial oxidative status. A group of 50 women from two Orthodox monasteries in Northern Greece and one group of 50 healthy lay women were included. During enrollment a detailed recording of dietary habits was performed, along with a scientific registry of demographic and anthropometric characteristics (via bioimped-ance).Orthodox nuns followed a typical Orthodox fasting regimen[daily feeding window (8 am–4 pm)] whereas lay women followed a TRE 16:8 regimen with the same feeding time-window with recommenda-tion to follow a low-fat diet, without characteristics of the Mediterranean diet. We included a complete biochemical analysis, as well as calciotropic profiles [Calcium-Ca, Albumin, Parathyroid hormone-PTH, 25-hydroxyvitamin D- 25(OH)D] as well as and markers of TAC (trichloroacetic acid), (glutathione) GSH and thiobarbituric acid reactive substances (TBARS) concentrations, as markers of oxidative status. All groups were comparable at baseline for calcium, PTH and 25(OH)D concentrations, with no significant differences between groups. Orthodox nuns manifested a lower median GSH compared to con-trols (6.0 vs. 7.2, p 0.04) and a higher median TAC (0.92 vs. 0.77, p <0.001). TBARS comparisons showed no significant difference between the two groups. No significant associations of oxidative status with 25(OH)D, PTH and markers of glucose homeostasis were evident. Results of this small pilot study indicate that both dietary regimens have advantages over oxidative markers compared to each other, with increased TAC in the groups of Orthodox Nuns after a 16th week period of COF compared to a 16:8 TRE and increased GSH concentrations in the lay women group. Future randomized trials are required to investigate superiority or non-inferiority between these dietary patterns, in the daily clinical setting.

Full text

Article Not peer-reviewed version
Effects of Religious Fasting on Markers
of Oxidative Status in Vitamin D
Deficient and Overweight Orthodox
Nuns versus Implementation of Time-
Restricted Eating in Lay Women from
Central and Northern Greece
Spyridon N Karras * , Konstantinos Michalakis , Fotios Tekos , Zoi Skaperda , Periklis Vardakas ,
Panayiotis D Ziakas , Maria Kypraiou , Marios Anemoulis , Antonios Vlastos , Georgios Tzimagiorgis ,
Konstantinos Chaitoglou , Neoklis Georgopoulos , Evangelos G. Papanikolaou , Demetrios Kouretas
Posted Date: 27 August 2024
doi: 10.20944/preprints202408.1910.v1
Keywords: oxidative stress; mediterranean diet; time restricted eating; orthodox fasting
Preprints.org is a free multidiscipline platform providing preprint service that
is dedicated to making early versions of research outputs permanently
available and citable. Preprints posted at Preprints.org appear in Web of
Science, Crossref, Google Scholar, Scilit, Europe PMC.
Copyright: This is an open access article distributed under the Creative Commons
Attribution License which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.


Article
EffectsofReligiousFastingonMarkersofOxidative
StatusinVitaminDDeficientandOverweight
OrthodoxNunsversusImplementationof
Time‐RestrictedEatinginLayWomenfromCentral
andNorthernGreece
KarrasSpyridon1,MichalakisKonstantinos2,TekosFotios3,SkaperdaZoi3,PeriklisVardakas3,
ZiakasD.Pa‐nayiotis4,KypraiouMaria5,AnemoulisMarios6,VlastosAntonios6,
TzimagiorgisGeorgios1,ChaitoglouKonstantinos1,GeorgopoulosNeoklis7andPapanikolaou
Evangelos5,KouretasDemetrios3
1LaboratoryofBiologicalChemistry,MedicalSchool,AristotleUniversity,55535Thessaloniki,Greece
2EndocrinePractice,DepartmentofObesityandMetabolism,Athens,Greece
3DepartmentofBiochemistry‐Biotechnology,SchoolofHealthSciences,UniversityofThessaly,
Larissa,Greece
4DepartmentofMedicine,UniversityofBrown,RI,USA
5AssistingNatureCentreofReproductionandGenetics,Thessaloniki,Greece
6MedicalSchool,AristotleUniversity,Thessaloniki,Greece
7DivisionofEndocrinology,DepartmentofInternalMedicine,SchoolofHealthSciences,Universityof
Patras,26504Patras,Greece
Abstract:Mediterraneandiethasbeenwidelysuggestedtoexertsignificantbeneficialeffectson
endothelialoxida‐tivestatusandcardiometabolichealth.GreekOrthodoxmonasteries,duetotheir
specificnutritionalandsartorialhabits,compriseapopulationwhichstrictlyadherestonutritional
patternswithrestrictedeatingandaplant‐basedsubsetoftheMediter‐raneandiet,often
accompaniedbyprofoundhypovitaminosisD.Time–restrictedeatingisalsoadoptedfromalarge
partofgenerallayGreekpopulationforhealthpromotingreasons,withoutrestrictionsonan‐imal
productconsumption,asimposedbyOrthodoxreligiousfasting.However,comparativeeffectsof
thesenutritionalpatternsonoxidativestressmarkersremainscarce.Thepresentstudyattempted
toevaluatetheeffectsofChristianOrthodoxfasting(COF)inagroupofvitaminD–deficientand
overweightOrthodoxnunsfromCentralandNorthernGreece,comparedtotheimplementationof
TRE,16:8dietaryregimeninacohortofadultwomenfromthegeneralpopulationfromthesame
region,withregardtomarkersofendothelialoxidativestatus.Agroupof50womenfromtwo
OrthodoxmonasteriesinNorthernGreeceandonegroupof50healthylaywomenwereincluded.
Duringenrollmentadetailedrecordingofdietaryhabitswasperformed,alongwithascientific
registryofdemographicandanthropometriccharacteristics(viabioimped‐ance).Orthodoxnuns
followedatypicalOrthodoxfastingregimen[dailyfeedingwindow(8am–4pm)]whereaslay
womenfollowedaTRE16:8regimenwiththesamefeedingtime‐windowwithrecommenda‐tion
tofollowalow‐fatdiet,withoutcharacteristicsoftheMediterraneandiet.Weincludedacomplete
biochemicalanalysis,aswellascalciotropicprofiles[Calcium‐Ca,Albumin,Parathyroidhormone‐
PTH,25‐hydroxyvitaminD‐ 25(OH)D]aswellasandmarkersofTAC(trichloroaceticacid),
(glutathione)GSHandthiobarbituricacidreactivesubstances(TBARS)concentrations,asmarkers
ofoxidativestatus.Allgroupswerecomparableatbaselineforcalcium,PTHand25(OH)D
concentrations,withnosignificantdifferencesbetweengroups.Orthodoxnunsmanifestedalower
medianGSHcomparedtocon‐trols(6.0vs.7.2,p0.04)andahighermedianTAC(0.92vs.0.77,p
<0.001).TBARScomparisonsshowednosignificantdifferencebetweenthetwogroups.No
significantassociationsofoxidativestatuswith25(OH)D,PTHandmarkersofglucosehomeostasis
wereevident.Resultsofthissmallpilotstudyindicatethatbothdietaryregimenshaveadvantages
Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and
contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting
from any ideas, methods, instructions, or products referred to in the content.
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1
© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

2
overoxidativemarkerscomparedtoeachother,withincreasedTACinthegroupsofOrthodox
Nunsaftera16thweekperiodofCOFcomparedtoa16:8TREandincreasedGSHconcentrations
inthelaywomengroup.Futurerandomizedtrialsarerequiredtoinvestigatesuperiorityornon‐
inferioritybetweenthesedietarypatterns,inthedailyclinicalsetting.
Keywords:oxidativestress;Mediterraneandiet;timerestrictedeating;Orthodoxfasting

Introduction
ChristianOrthodoxfasting(COF),isavitalsubsetoftheMediterraneandiet(MD)
[1–4],whichforreligiousreasonsisconsideredtobedeeplyintegratedintheculturaldietary
behaviorofalargepartoftheGreekpopulation[5–9]forprolongedperiods(from120to180d)
annually[8].Orthodoxmonasteriesfollowthisarchetypepatternofdietthroughouttheyear,with
periodsofmorestrictfastingrituals2‐6weeksbeforereligiouscelebrations,asawayofphysicaland
mentalprosperityandpersonalspiritualdevelopment[1,2].However,besidesthespiritual
significanceofCOF,aplethoraofcohortstudiessuggestthatCOFsharesthebeneficialeffectsofthe
typicalMDbypromotingspecificcardioprotectivemechanisms,includingreducedintakeofdietary
cholesterolandfattyacids,thusprovidingoptimaleffectsonplasmalipidconcentrations[9,10].
Thesebenefitshavebeenmainlyattributedtotheintegrationofaplant‐baseddietalongwith
characteristicsofdietaryrestrictionofanimalproducts(meat,dairyproductsandeggs)[6]and
restrictionofcaloricintakeduringCOFperiods[11–14].Wehavepreviouslyreportedonthe
beneficialeffectsofCOFontheadipokineprofile[7,8,14]aswellasonglucosehomeostasisinboth
monasticandgeneralpopulations[15],asmarkersforpreventionofcardiovasculardyshomeostasis,
withtheexceptionofprofoundhypovitaminosis—DinOrthodoxMonks,mainlyduetotheir
sartorialhabits[11,16].
Additionally,restrictionoffoodintakeinspecifictime—framesduringtheday,hasalsobeen
hypothesizedtocontributetothebenefitsdescribedabove,acharacteristicwhichattractedsignificant
scientificandpublicinterestduringthelastdecade,throughvariousintermittent—fastingpatterns,
practicedworldwideasahealth‐promotingdiet[17].Time‐restrictedeating(TRE),includesspecific
time‐framesoffoodintakeduringtheday,whichvaryfrom4‐12hoursdaily[e.g.,20hoursoffasting
vs4hoursofpermittedfoodintake—20:4—aswellasadditionaltimeframes(18:6,16:8etc.)][18].On
theotherhand,impairmentofantioxidativecapacityofvascularendotheliumisanestablished
aggravatingfactorfordevelopmentofendothelialdysfunctionandfuturecardiovascularmajor
events[19,20].Onthatbasis,aconsiderablenumberofpreviousstudies[21–24]havesuggestedthat
MDisstronglyassociatedwithfavorableeffectsonoxidativestatus,implyingapotentialpathway
forexertingitswell—establishedcardiovascularbenefits.However,resultsonCOFasavitalsubset
ofMDanditseffectsonoxidativestatus,particularlycomparedtootherhealthynutritionalpatterns
widelyadoptedbythegeneralpopulation,remainscarce.
Additionally,thesepotentialinteractionshavesofarnotbeeninvestigatedinconjunctionwith
othermetabolicconditionsassociatedwithendothelialdysfunction,includingimpairmentofvitamin
Dstatusandinsulinresistance,particularlyinvitaminDdeficientandoverweightindividuals.These
resultscouldelucidatepotentialmechanismsofMD—relatedeffectsonantioxidativecapacityand
alsoelaborateontheresearchhypothesis,whichindicatesthemacro‐andmicronutrientsynthesis
andincreasedintakeoffoodantioxidants,ratherthanthetimingoffoodintake,asthecornerstoneof
attainedmetabolicbenefits.
ThepresentstudyattemptedtoevaluatetheeffectsofCOFinagroupofvitaminD–deficient
andoverweightOrthodoxnunsfromCentralandNorthernGreece,comparedtotheimplementation
ofTRE16:8dietaryregimeninacohortofadultwomenfromthegeneralpopulationfromthesame
region,withregardtomarkersofendothelialoxidativestatus.

Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

3
Methods
Design
Thiswasacross‐sectionalstudyafteraperiodof16‐weekimplementationofCOFandTRE,in
twogroupsofadultfemalenunsandlaywomen.
StudyPopulation
Weincluded50ChristianOrthodoxfemaleadultnuns,fromtwodifferentmonasteries,30–50
yearsofage,residingintheCentralandNorthernGreeceandanage—matchedcohortof50adult
laywomenfromthesameregion.
Orthodoxnuns(butnotlaywomen),withabaseline25‐hydroxyvitaminDconcentrations≥20
ng/ml(asinitiallyevaluatedfromthesameinitialcohort–resultspublishedpreviously[12–15])were
excluded.Additionalexclusioncriteriaforbothgroupswere:bodymassindex(BMI)≤ 25,
amenorrhea≥3months,pregnancy,presenceofchronickidneydisease,severeliverdisease,
diagnosisofprediabetes(fastingglucose100–125mg/dLorglycatedhemoglobin5.7–6.4%orblood
glucose140–199mg/dLat2hpost75gglucoseload)ordiabetesmellitus(fastingglucose≥126mg/dL
orglycatedhemoglobin≥6.5%orbloodglucose≥200mg/dLat2hpost75gglucoseload),
dyslipidemia,arterialhypertension,oruncontrolledhypothyroidism(notadequatelycontrolledor
firstdiagnosedandnottreated),(recentsurgeryorsevereinfections(duringthepast3months),
administrationofmedicationsthatcanalterbodyweight,glucoseandlipidmetabolism(e.g.,statins,
corticosteroids,antipsychotics),intakeofvitaminsormineralsupplements,physicaldisabilities
and/orneurodegenerativedisordersthatcouldaffectphysicalactivity,acuteinfectionsandchronic
degenerativediseases.
DietaryPatterns
Orthodoxnunswithatleast16weeksadherencetoCOFwereincludedinthestudy,whereas
womenfromthegeneralpopulation,followedTREfor16weeks,afterawash‐outperiodof3weeks,
beforeinclusioninthestudy.OrthodoxnunsfollowedtheAthoniantypeoffastingaspreviously
described[1–4],abstainingfromconsumptionofanimalproducts(meat,poultry,eggs,dairyand
cheese),withtheexceptionofseafoodandfish,whichfasterswerepermittedtoeatontwospecific
weekdays,whilethegeneralpopulationgroupwasallowedtoeatlow‐fatmeatproducts,without
specificdistributionandcut‐offsofmacronutrientsanddailycaloricintake.
Orthodoxnunsgroupadoptedan8heatinginterval(08.00to16.00),asdictatedbytypical
monasterydietaryrules,whichareobligatoryforallresidentsofthemonastery,whileTREgroup
consumedfoodfrom09:00to17:00.Adherencetodietaryplanswasevaluatedwitha3‐dayfood
record(twoweekdaysandoneweekendday)attheendofthestudyperiod,whiletheNutrition
AnalysisSoftwareFoodProcessor[https://esha.com/products/food‐processor/(accessedon2August
2024)][25]wasusedtoanalyzefoodrecords.Finally,levels,frequencyanddurationofphysical
activity,dividedinlight,moderateandintensephysicalactivity,wererecordedforallparticipants,
accordingtoAHArecommendations[26].
AnthropometricMeasurementsandBiochemicalAnalysis
Anthropometricmeasurementsandbiochemicalanalyseswereperformedinbothgroupsusing
standardizedprocedures.Exactmethods,referenceranges,equipmentused,andotherdetailswere
previouslyanalyticallydescribed[11].Inbrief,bodyweight(BW)wasrecordedtothenearest0.01kg
usingacalibratedcomputerizeddigitalbalance(K‐TronP1‐SR,OnrionLLC,Bergenfield,NJ,USA);
eachparticipantwasbarefootandlightlydressedduringmeasurement.BMIwascalculatedasthe
ratioofweightinkilogramsdividedbytheheightinmeterssquared(kg/m2)[27].Bodyfat(BF)mass
andpercentage,visceralfat(VF),musclemass,fat‐freemass,andtotalbodywaterweremeasured
usingbioelectricalimpedanceanalysis(SC‐330S,TanitaCorporation,Tokyo)[28].Bloodsamples
weredrawninthemorning,aftera12hovernightfastbyantecubitalvenipuncture,andthesamples
werestoredat−20oCpriortoanalysis.Calcium(Ca)concentrationswereevaluatedusingthe
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

4
COBAS8000automatedanalyzersystem(RocheDiagnosticsGmbH,D‐68298Mannheim,Germany).
Parathyroidhormone(PTH)and25(OH)D,weretestedintheCOBASe602immunochemistry
moduleusingelectro‐chemiluminescence(ECL)technology(RocheDiagnosticsGmbH,D‐68298
Mannheim,Germany).Referencerangesofvaluesaswellasinter‐ andintra‐assaycoefficientsof
variationfortheexaminedparametersareasfollows:Ca:8.4–10.2mg/dl,0.8–1.3%and0.5–1.3%;PTH:
15–65pg/mL(or1.6–6.9pmol/L),1.1–2.0%and2.5–3.4%;25(OH)D:≥30ng/mL,2.2–6.8%and3.4–
13.1%.Insulinresistancewascalculatedusingthehomeostasismodelassessment(HOMA‐IR)
formuladescribedbyMatthewsetal.[29]asfollows:FPI(mU/mL)xFPG(mmol/L)/22.5,whereFPI
standsforfastingplasmainsulinandFPGforfastingplasmaglucose.
MarkersofOxidativeStatus
DeterminationofGlutathione(GSH)ConcentrationinBlood
GSHconcentrationwasdeterminedaccordingtothemethodofReddyetal.[30]aspreviously
described[31].Atfirst,400μLofRBCLwasmixedwith400μLof5%trichloroaceticacid(TCA),
respectively,andcentrifuged(15,00×g,5min,5°C).Afterwards,300μLofthesupernatantwasmixed
with90μLof5%TCAandcentrifuged(15,00×g,5min,5°C).Theresultingsupernatantwascollected
andusedasthebiologicalsamplefortheassay.Regardingtheassay,20μLofthebiological
samplewasmixedwith660μLofphosphatebuffer(67mM,pH=7.95)and330μLof5,5‐dithiobis(2‐
nitrobenzoicacid)(DTNB)(1mM).Thesampleswerevortexedandincubatedfor45mininthedark
atroomtemperature(RT),andtheopticaldensitywasmeasuredat412nm.GSHconcentrationwas
calculatedbasedonthemillimolarextinctioncoefficientof2‐nitro‐5‐thiobenzoate(TNB)(13.6
L/mmol/cm).
DeterminationofTotalAntioxidantCapacity(TAC)ConcentrationsinBlood
TAClevelswereevaluatedbasedontheprotocolofJanaszewskaandBartosz[32].More
elaborately,20μLofplasmawasmixedwith480μLor460μLofphosphatebuffer(10mM,pH=7.4),
respectively,and,immediately,500μLof2,2‐diphenyl‐1‐picrylhydrazylradical(DPPH•)solution
(0.1mM)wasadded.Thesampleswerevortexed,incubatedfor1hinthedarkatRT,andcentrifuged
(15,00×g,3min,25°C).Finally,theopticaldensitywasmeasuredat520nm.TAClevelswere
expressedasthemmolofDPPH•reducedtothecorrespondinghydrazinebytheantioxidant
compoundspresentinplasmaortissuehomogenates.
DeterminationofThiobarbituricacidReactiveSubstances(TBARS)ConcentrationsinBlood
TBARSlevelsweredeterminedbyaslightlymodifiedmethodbyKelesetal.[33].Specifically,
100μLofplasmawasmixedwith500μLofTris‐HCl(200mM,pH=7.4)and500μLof35%TCAand
incubatedfor10minatRT.Afterthat,1mLofsodiumsulfate(Na2SO4)(2M)andthiobarbituricacid
(TBA)(55mM)solutionwasadded,andthesampleswereplacedinawaterbathfor45minat95°C.
Afterincubation,thesampleswerecooledonicefor5min,1mLof70%TCAwasadded,andthe
sampleswerecentrifuged(11,20×g,3min,25°C).Theresultingsupernatantwasusedtomeasurethe
opticaldensityat530nm.TBARSlevelswerecalculatedbyapplyingthemolarextinctioncoefficient
ofmalonyldialdehyde(ΜDA)(156,000L/mol/cm).
DeterminationofGSHConcentrationinBlood
EthicalConsiderations
ThestudywasconductedinaccordancewiththeDeclarationofHelsinkionthehumantrial
performance.Writteninformedconsentforinclusioninthestudywasgivenbyparticipants.Official
writtenapprovalfortheinclusionoftheOrthodoxnunsgroupwasgivenbytheHolySupervision
Councilofthemonasteries,aftersubmissionofthefullstudyprotocol12monthsbeforestudy
initiation.

Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

5
StatisticalAnalysis
ContinuousvariablesarereportedasmeansandS.D.’s.Dietaryandnutrientintakeonwere
comparedusingpairedsamplest‐test.Agedifferencesbetweenthegroupswithlight,moderateand
intensephysicalactivityweretestedusingone‐wayanalysisofvariancewithTukeyposthoctest.
Theeffectoflevelofphysicalactivityonoverallhealthmarkerswastestedwithanalysisofcovariance
tocontrolforage.NormalityofdistributionwastestedwithonesampleKolmogorov–Smirnovtest
(exactstatistics).
Theamong‐groupcomparisonwasmadeusingnonparametricMann‐WhitneyUtest.Linear
regressionwasusedformulti‐adjustedanalysis.Assumptionswerecheckedforeachstatistical
analysis.LevelofsignificancewassetatP<0.05(non‐directional).DatawereanalyzedusingSPSS
v22.
Results
Orthodoxnunswereolderthanlaywomen(medianage42vs.38,p<0.001)butdidnotdifferin
medianweightandBMI(Table1).Groupsdidnotdifferinbodyfat(%),leanbodymass(%)andwaist
circumference,aswellasdegreesofphysicalactivity,withtheexceptionofintenseactivity,inwhich
laywomenreportedhigherrates.Regardingnutritionalanalysis,laywomenconsumedhigher
amountsofcarbohydrates(gr)(194.3±23.4vs159.6±21.8),totalandsaturatedfat(24.4±0.6vs21.0±
0.1and16.4±0.0and12.7±0.0,respectively)whereasOrthodoxnunsreportedhigheramountsof
proteinandfibreintake(36.1±0.8vs24.2±0.8).
Althoughexpected,accordingtothestudyprotocol,hypovitaminosisDevidentintheOrthodox
nuns’group,resultedinsignificantlyhighermedianserumPTHthanamonglaywomen(45.6vs.
19.4,p<.001),afteradjustingforseasonalvariation.Inaddition,afteradjustingforageand25(OH)D3
concentrationsinlinearregressionacrossallpatients,PTHhadasignificantpositiveassociationwith
age(+6.0pg/mlper10‐yearincreaseinage,p<0.001)andasignificantnegativeassociationwithserum
25(OH)D3status(–0.61pg/mlperng/mlincreaseinserumD3).Orthodoxnunsdemonstratedlower
medianfastinginsulinconcentrations(5.3vs.7.2,p0.02)comparedtolaywomenandevenafter
adjustingforageandBMI,thedifferenceremainedsignificant;Ofmajorinterestisthefact,that
insulinconcentrationslackedasignificantassociationwithBMIorageinbothgroups.Regarding
redoxstatus,OrthodoxnunsmanifestedalowermedianGSHcomparedtocontrols(6.0vs.7.2,p.04)
andahighermedianTAC(0.92vs.0.77,p<.001).TBARScomparisonsshowednosignificant
differencebetweenthetwogroups.Afteradjustingforageinlinearregression,Orthodoxnunshad
alowerGSHconcentrationinserum(meandifference‐1.7;95%CI‐2.7to=–0.7,p.001)comparedto
controls,whiletheageeffectwasnotsignificant(p=0.45).Afteradjustingforage,nunshadahigher
TACconcentrationinserum(meandifference0.19;95%CI0.13to0.26,p<.001),whereasafter
adjustingforage,BMIandtotalfatinlinearregression,nunshadalowerGSHconcentrationinserum
(meandifference–1.6;95%significant(p=0.45).Afteradjustingforage,nunshadahigherTAC
concentrationinserum(meandifference0.19;95%CI0.13to0.26,p<.001),whereasafteradjustingfor
age,BMIandtotalfatinlinearregression,nunshadalowerGSHconcentrationinserum(mean
difference–1.6;95%CI–2.6to=–0.7,p.001)comparedtolaywomen,whiletheageandBMIeffects
werenotsignificant.AfteradjustingforageandBMI,nunshadahigherTACconcentrationinserum
(meandifference0.21;95%CI0.15to0.27p<.001);age,BMIandtotalfat,effectswerenotsignificant.
Nosignificantassociationsofoxidativestatuswith25(OH)D,PTHandmarkersofglucose
homeostasiswereevident.
Table1.
Orthodoxnuns(n=50)Laywomen(n=50)p
Demographics  
Age(years)42(36‐50)38(34‐42)0.03
Wei ght(kg)71.5(64‐82)66(60‐87)0.31
BMI(kg/m2)27.0(24.2‐29.0)26.8(22.0‐32.0)0.19
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

6
Bodyfat(%)24.5±9.422.1±8.10.23
Leanbodymass
(%)39.9±6.341.2±7.10.15
Wai s t
circumference
(cm)
92.489.10.11
Physicalactivity
LightN=9N=70.31
ModerateN=27N=250.48
IntenseN=14N=180.03
Yea r s of
monasticism10.5±9.8‐‐
Deaconshipof
OrthodoxNuns
Baker(3);Botanist(2);Cook(5);Cookingassistant(5);Diningassistant(5);
Ecclesiasticalchanter(6);Gardener(3);Housekeeper(3);Iconographer(6);
Laundryassistant(4);Pharmacist(2);
Table2.
Energy(kcal)1565.9±64.51890.0±71.0<0.01
Carbohydrates(g)159.6±21.8194.3±23.40.03
Protein(g)89.2±1.372.3±1.30.04
Dailyfatintake(g)21.0±0.124.4±0.60.02
Dailysaturatedfatintake(g)12.7±0.016.4±0.00.01
Totalfibreintake(g)36.1±0.824.2±0.80.02
25‐hydroxy‐vitaminD3(ng/Ml)15.7(11.4‐19.8)26.1(18.2‐31.9)0.02
PTH(pg/ml)45.6(39.6‐54.7)19.4(13.1‐28.5)<0.001
Calcium(mg/dl)9.4(9.1‐9.7)9.1(8.8‐9.3)0.15
Insulin(IU/L)5.3(3.4‐6.7)7.1(4.7‐11)0.02
Fastingglucose(mg/dl)84.4±10.189.2±9.70.43
HOMA‐IR1.02±0.41.26±0.70.21
Oxidativestatus
TAC0.93(0.87‐0.99)0.77(0.65‐0.90)<.001
GSH6.0(4.4‐6.8)7.2(5.5‐8.8)0.04
TBARS7.3(5.8‐8.3)7.6(6.9‐8.4)0.28

Figure1.ConcentrationsofTACinOrthodoxNunsandlaywomengroup.
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

7

Figure2.ConcentrationsofGSHinOrthodoxNunsandlaywomengroup.
Discussion
Toourknowledge,thisthefirstcross‐sectionalstudyreportingpreliminaryresultsonthe
comparativeeffectsofCOFonoxidativestatusinvitaminDdeficientGreekOrthodoxnunsandTRE
(16:8)dietaryregimeninagroupoflaywomenwithvitaminDsufficiency.Theseresultsindicated:
i)increasedantioxidativecapacity(TAC)inthegroupofOrthodoxNunsaftera16weekperiodof
COFcomparedtoa16:8TRE,ii)increasedGSHlevelsinthelaywomengroupcomparedtothegroup
ofOrthodoxnunsaswellascomparableofTBARSlevelsinbothgroups,afteradjustingforseveral
confounders,whichsuggestpotentialdiverseeffectsofCOFandTREonoxidativestatus.
MDisaplant‐baseddiet,richinfruit,vegetables,nuts,herbs,withfewerfishanddairyproducts
andwithlessredmeatandredwine.MDincludesvariousnutritionalcompounds,withwell‐
establishedbeneficialeffectsonoxidativestatus.Aplethoraofpreviousbasicandclinicalstudies
suggestedthatMDhasbeenshowntobeoneofthehealthiesteatingpatterns,withvariousmetabolic
benefits,partlymediatedthroughitsantioxidantcapacity[34,35].Daietal.studiedtheratioof
reducedtooxidizedglutathione(GSH/GSSG)intwins.Thehighertheratio,thelowertheoxidative
stress,givingaresultofahigherratioupto7%inindividualswhofollowedtheMediterraneandiet,
regardlessoftheadjustmentoftheenergyintake[36].Inasub‐cohortofThePREDIMEDtrial,
participantswithhighcardiovascularriskwererandomizedtoaMediterraneandietsupplemented
withextra‐virginoliveoilandmanifestedasignificantreductionincellularlipidlevelsandlipid
oxidation,aswellasmalondialdehydeconcentrationsinmononuclearcells,withoutchangesin
serumglutathioneperoxidaseactivity[37].
DocumentedbenefitsofMDincludeconsumptionofunsaturatedfattyacids,foundinoliveoil,
whichcontain3,3dimethyl‐1‐butanol,thuspreventingtheformationoftrimethylamine‐1‐oxide,one
oftheoxidantsrelatedtocardiovascularevents[38,39].
Additionally,MDsynthesisisrichinoleic‐acidandalpha‐linoleic‐acid,foundinnuts,fruitand
vegetableflavonoids,aswellasomega‐3‐polyunsaturated‐fatty‐acids,andfiberandpolyphenols,all
ofwhichhaveanti‐oxidative,anti‐bacterialandanti‐inflammatoryeffects[40–42].Moreover,whole‐
grains,asavitalcompoundofMD,containapolyaminecalledspermidine,whichhasbeenshownto
extendchronologicallife‐spaninflies,nematodes,rodents,andhumancells.Spermidineisknownto
inhibithistoneacetyltransferases,whichresultsinhigherresistancetooxidativestress[43].
TREhasbeenalsotheobjectiveofrecentstudiesregardingitspotentialbeneficialeffectson
cardiometabolichealth.Giventhefactthathormonesundergoacircadianrhythm,metabolicand
stresshormonesasinsulin,cortisol,growthhormoneandmelatoninundergothesamevariation,
givingdifferentlevelsbetweenacalorie‐restrictingdietandintermittent‐fastingdiet,whichrestricts
thefeedingtimeincertainhours[44].McAllisteretal.studiedtheimpactofintermittentfastingon
markersofcardiometabolichealth,measuringseveralmarkersofinflammation,OS,and
cardiometabolichealth(insulin,ghrelin,leptin,glucagon,adiponectin,resistin,advancedglycated‐
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

8
endproducts(AGE),advancedoxidationproteinproducts,totalnitrite‐nitratelevels,tumornecrosis
factor‐α,interleukin(IL)‐6,IL‐8,IL‐10andshowedthattime‐restrictedfeedingresultedinsignificant
reductionsinadvancedoxidationproteinproducts(∼31%)andAGEs(∼25%);however,noother
changeswerefound[45].Recentrandomizedclinicaltrialsalsodemonstratedthata6‐hrfeeding
periodfor5weeksimprovedinsulinsensitivity,β‐cellresponsivenessandoxidativestress,
irrespectiveofweightloss[46].Theseresultswerealsopreviouslyconfirmedbyother
groups,where4‐and6‐hTREfor5weeks,resultedinareductionof8‐isoprostane,asamarkerof
oxidativestresstolipids,4‐hydroxynonenaladducts,proteincarbonylsandnitrotyrosine[47].
COFisaplant‐basedsubsetofthetraditionalMDfollowedformorethanathousandyearsfrom
alargepartoftheGreekOrthodoxgeneralpopulationforreligiouspurposesfrom90–150daysper
year.GreekOrthodoxmonasteriesadheretothisdietaryregimenthroughouttheyear,withthe
additionofTRE(usually16:8)characteristicsintheirdailydietaryregimen,whichisstrictlyfollowed
byallmembersofthemonasterialcommunity,comprisinganoptimalsamplefornutritionalstudies
.WehaverepeatedlyreportedontheeffectsonCOFonbodyweight,lipidparameters,adipokines
andvitaminDstatus,regardingtheexistenceofseverehypovitaminosisDinOrthodoxmalemonks,
mainlyduetotheirsartorialhabits.
However,thisisthefirstreportontheeffectsofCOFonoxidativeequilibrium,particularly
comparedtoahealth–promotingpatternlikeTRE.
Ourresearchhypothesisraisedthequestionfornon‐inferiorityofTREcomparedtoCOF,ina
vitaminDdeficientmonasticpopulation(asmostsimilarmonasticcommunitiesinGreece),taking
intoaccountthatwomenincludedintheTRE,werenotinstructedtofollowaMD—specificdietary
pattern.Accordingtopreviousresults,chronicvitaminDdeficiencyisastateofincreasedoxidative
stress,whichreducesthecapacityofmitochondrialrespiration,throughmodulatingnuclearmRNA
downregulatingtheexpressionofcomplexIoftheelectrontransportchain,reducingofadenosine
triphosphate(ATP),resultinginincreasedformationofROS,augmentingoxidativestress[48].
Maintainingoptimumlevelsofredoxbiomarkersiscrucialforpreventingoxidativedamage,
supportingdetoxificationprocesses,andensuringproperimmunefunction.Previousliterature
proposedthatclusteringofhighandlowGSHlevelsmightprovidestrongcausalityfortype2
diabetesandmetabolicsyndrome[48].OurresultsfailedtosuggestasuperiorityofCOFoverTRE,
inagroupwithconfirmedMD‐typedietaryregimenandTREcharacteristicsasOrthodoxnuns,
comparedtoa16:8withoutspecificMD—relateddietarycharacteristics.
AplausibleexplanationcouldbethatthegeneralpopulationfollowingaTREpatterncomply
withahealthydietarypattern,whichdespitenotbeingidenticaltoMD,alsoexertsbenefitsonGSH
concentrations,alwaystakingintoaccountthelimitationsofthisstudy.Anotherexplanation,could
lieonthepotentialadverseeffectsofhypovitaminosisD,evidentinOrthodoxnunsincludedinthis
study,onGSHconcentrations,aspreviouslyreported[12].VitaminDsupplementationinthisgroup
ofvitaminDdeficientnunscouldelucidatethispotentialbiologicalassociationonGSHstatus.TRE
couldalsohaveindependentbeneficialeffectsonoxidativestatus,whichareevidentwithoutstrict
adherencetoaMD‐relatedpattern,aspreviouslyreported[12].Finally,ourstudyfailedtoestablish
anassociationofimpairedvitaminDstatusandoxidativemarkers,whichcouldbeattributedtoits
cross‐sectionaldesign.Thisstudyhasseverallimitationsandcanonlybeconsideredasapilotstudy,
withfindingswhichdefinitelyrequireconfirmationinaprospectivestudy.
Indetail,thenumberofincludedparticipantswasrelativelysmall;however,thisisa
representativesampleofOrthodoxnuns,accordingtotheirdietaryandphysicalactivityplan.We
havealsonotincludedadetailedanalysisregardingtheintakeofthedietaryantioxidativesinthe
twogroups,whichcouldexplaindiversityinmarkersofoxidativestatus.Finally,sincenobaseline
evaluation,priortotheimplementationofdietaryinterventions,wasfeasibleforbothgroups,we
wereunabletoestablishcausalassociations.
Inconclusion,resultsofthissmallpilotstudyindicatethatbothdietaryregimenshave
advantagesoveroxidativemarkers,comparedtoeachother,withincreasedTACinthegroupsof
OrthodoxNunsaftera16th‐weekperiodofCOFincomparisontoincreasedGSHconcentrationsin
thelaywomengroupfollowing16:8TRE,andcomparableconcentrationsofTBARS.Future
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

9
randomizedtrialsarerequiredtoinvestigatesuperiorityornon‐inferioritybetweenthesedietary
patterns,inthedailyclinicalsetting.
References
1. Trepanowski,J.F.;Bloomer,R.J.Theimpactofreligiousfastingonhumanhealth.Nutr.J.2010,9,57.
2. Sarri,K.O.;Linardakis,M.K.;Bervanaki,F.N.;Tzanakis,N.E.;Kafatos,A.G.GreekOrthodoxfastingrituals:
AhiddencharacteristicoftheMediterraneandietofCrete.Br.J.Nutr.2004,92,277–284.
3. Sarri,K.O.;Tzanakis,N.E.;Linardakis,M.K.;Mamalakis,G.D.;Kafatos,A.G.EffectsofGreekOrthodox
ChristianChurchfastingonserumlipidsandobesity.BMCPublicHealth2003,3,16.
4. Sarri,K.;Linardakis,M.;Codrington,C.;Kafatos,A.DoestheperiodicvegetarianismofGreekOrthodox
Christiansbenefitbloodpressure?Prev.Med.2007,44,341–348.
5. Karras,S.N.;Koufakis,T.;Adamidou,L.;Antonopoulou,V.;Karalazou,P.;Thisiadou,K.;Mitrofanova,E.;
Mulrooney,H.;Petróczi,A.;Zebekakis,P.;etal.Effectsoforthodoxreligiousfastingversuscombined
energyandtimerestrictedeatingonbodyweight,lipidconcentrationsandglycaemicprofile.Int.J.Food
Sci.Nutr.2021,72,82–92.
6. Karras,S.N.;Koufakis,T.;Adamidou,L.;Polyzos,S.A.;Karalazou,P.;Thisiadou,K.;Zebekakis,P.;
Makedou,K.;Kotsa,K.Similarlateeffectsofa7‐weekorthodoxreligiousfastingandatimerestricted
eatingpatternonanthropometricandmetabolicprofilesofoverweightadults.Int.J.FoodSci.Nutr.2021,
72,248–258.
7. Karras,S.N.;Koufakis,T.;Adamidou,L.;Dimakopoulos,G.;Karalazou,P.;Thisiadou,K.;Makedou,K.;
Kotsa,K.EffectsofChristianOrthodoxFastingVersusTime‐RestrictedEatingonPlasmaIrisin
ConcentrationsamongOverweightMetabolicallyHealthyIndividuals.Nutrients2021,13,1071.
8. Karras,S.N.;Koufakis,T.;Adamidou,L.;Dimakopoulos,G.;Karalazou,P.;Thisiadou,K.;Makedou,K.;
Zebekakis,P.;Kotsa,K.ImplementationofChristianOrthodoxfastingimprovesplasmaadiponectin
concentrationscomparedwithtime‐restrictedeatinginoverweightpremenopausalwomen.Int.J.Food
Sci.Nutr.2022,73,210–220.
9. Azzeh,F.S.;Hasanain,D.M.;Qadhi,A.H.;Ghafouri,K.J.;Azhar,W.F.;Ghaith,M.M.;Aldairi,A.F.;
Almasmoum,H.A.;Assaggaf,H.M.;Alhussain,M.H.;etal.ConsumptionofFoodComponentsofthe
MediterraneanDietDecreasestheRiskofBreastCancerintheMakkahRegion,SaudiArabia:ACase‐
ControlStudy.Front.Nutr.2022,9,863029.
10. Trichopoulou,A.;Vasilopoulou,E.;Georga,K.Macro‐andmicronutrientsinatraditionalGreekmenu.
Forum.Nutr.2005,57,135–146.
11. Karras,S.N.;Koufakis,T.;Petróczi,A.;Folkerts,D.;Kypraiou,M.;Mulrooney,H.;Naughton,D.P.;
Persynaki,A.;Zebekakis,P.;Skoutas,D.;etal.ChristianOrthodoxfastinginpractice:Acomparative
evaluationbetweenGreekOrthodoxgeneralpopulationfastersandAthonianmonks.Nutrition2019,59,
69–76.
12. Karras,S.N.;Persynaki,A.;Petróczi,A.;Barkans,E.;Mulrooney,H.;Kypraiou,M.;Tzotzas,T.;Tziomalos,
K.;Kotsa,K.;Tsioudas,A.;etal.HealthbenefitsandconsequencesoftheEasternOrthodoxfastingin
monksofMountAthos:Across‐sectionalstudy.Eur.J.Clin.Nutr.2017,71,743–749.
13. 13Karras,S.N.;Koufakis,T.;Adamidou,L.;Dimakopoulos,G.;Karalazou,P.;Thisiadou,K.;Zebekakis,P.;
Makedou,K.;Kotsa,K.Differentpatternsofchangesinfree25‐hydroxyvitaminDconcentrationsduring
intermittentfastingamongmeateatersandnon‐meateatersandcorrelationswithaminoacidintake.Int.
J.FoodSci.Nutr.2023,74,257–267.
14. KarrasSN,KoufakisT,PopovicDS,AdamidouL,KaralazouP,ThisiadouK,ZebekakisP,MakedouK,
KotsaK.Nutrients.2023Dec9;15(24):5058.doi:10.3390/nu15245058AMediterraneanEatingPattern
CombiningEnergyandTime‐RestrictedEatingImprovesVaspinandOmentinConcentrationsCompared
toIntermittentFastinginOverweightIndividuals.
15. KarrasSN,KoufakisT,DimakopoulosG,PopovicDS,KotsaK.Changesindietaryintakeofasparticacid
duringandafterintermittentfastingcorrelatewithanimprovementinfastingglucoseinoverweight
individuals.JDiabetes.2023Feb;15(2):181‐184.doi:10.1111/1753‐0407.13351.CrossRef]
16. KarrasSN,APersynaki,APetróczi,EBarkans,HMulrooney,MKypraiou1,TTzotzas,KTziomalos,K
Kotsa,ATsioudas,CPichard,DPNaughtonHealthbenefitsandconsequencesoftheEasternOrthodox
fastinginmonksofMountAthos:across‐sectionalstudyEurJClinNutr.2017Jun;71(6):743‐749.doi:
10.1038/ejcn.2017.26.Epub2017Mar22.
17. ManoogianENC,LaferrèreBTime‐restrictedeating:Whatweknowandwherethefieldisgoing..Obesity
(SilverSpring).2023Feb;31Suppl1(Suppl1):7‐8.doi:10.1002/oby.23672.
18. KoppoldDA,BreinlingerC,HanslianE,KesslerC,CramerH,KhokharAR,PetersonCM,TinsleyG,
VernieriC,BloomerRJ,BoschmannM,BragazziNL,BrandhorstS,GabelK,GoldhamerAC,Grajower
MM,HarvieM,HeilbronnL,HorneBD,KarrasSN,LanghorstJ,LischkaE,MadeoF,MitchellSJ,
Papagiannopoulos‐VatopaidinosIE,PapagiannopoulouM,PijlH,RavussinE,Ritzmann‐WidderichM,
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

10
VaradyK,AdamidouL,ChihaouiM,deCaboR,HassaneinM,LessanN,LongoV,ManoogianENC,
MattsonMP,MuhlesteinJB,PandaS,PapadopoulouSK,RodopaiosNE,StangeR,Michalsen
A.Internationalconsensusonfastingterminology.CellMetab.2024Aug6;36(8):1779‐1794.e4.doi:
10.1016/j.cmet.2024.06.013.Epub2024Jul25.
19. WangX,HeB.Endothelialdysfunction:molecularmechanismsandclinicalimplications.MedComm
(2020).2024Jul22;5(8):e651.doi:10.1002/mco2.651.eCollection2024Aug.
20. SharebianiH,MokaramM,MirghaniM,FazeliB,StanekA.TheEffectsofAntioxidantSupplementation
onthePathologicMechanismsofMetabolicSyndromeandCardiovascularDisease
Development..Nutrients.2024May27;16(11):1641.doi:10.3390/nu16111641.
21. DobroslavskaP,SilvaML,VicenteF,PereiraPMediterraneanDietaryPatternforHealthyandActive
Aging:ANarrativeReviewofanIntegrativeandSustainableApproach.Nutrients.2024May
31;16(11):1725.doi:10.3390/nu16111725.
22. RezigL,GhzaielI,KsilaM,YammineA,NuryT,ZarroukA,SamadiM,ChouaibiM,VejuxA,LizardG
CytoprotectiveactivitiesofrepresentativenutrientsfromtheMediterraneandietandofMediterraneanoils
against7‐ketocholesterol‐and7beta‐hydroxycholesterol‐inducedcytotoxicity:Applicationtoage‐related
diseasesandcivilizationdiseases..Steroids.2022Nov;187:109093.doi:10.1016/j.steroids.2022.109093.Epub
2022Aug24.
23. KhalilM,ShanmugamH,AbdallahH,JohnBrittoJS,GaleratiI,Gómez‐AmbrosiJ,FrühbeckG,Portincasa
PThePotentialoftheMediterraneanDiettoImproveMitochondrialFunctioninExperimentalModelsof
ObesityandMetabolicSyndrome..Nutrients.2022Jul28;14(15):3112.doi:10.3390/nu14153112.
24. OliveiraJS,daSilvaJA,deFreitasBVM,AlfenasRCG,BressanJ.AMediterraneandietimprovesglycation
markersinhealthypeopleandinthosewithchronicdiseases:asystematicreviewofclinicaltrials.Nutr
Rev.2024May8:nuae045.doi:10.1093/nutrit/nuae045.Onlineaheadofprint.
25. GreekNationalDietaryGuidelinesforAdults.Availableonline:
http://www.fao.org/nutrition/education/food‐ dietary‐ guidelines/regions/countries/greece/en/(accessed
on25July2024).
26. Jensen,M.D.;Ryan,D.H.;Apovian,C.M.;Ard,J.D.;Comuzzie,A.G.;Donato,K.A.;Hu,F.B.;Hubbard,V.S.;
Jakicic,J.M.;Kushner,R.F.;etal.2013AHA/ACC/TOSguidelineforthemanagementofoverweightand
obesityinadults:AreportoftheAmericanCollegeofCardiology/AmericanHeartAssociationTaskForce
onPracticeGuidelinesandTheObesitySociety.Circulation2014,129,S102–S138.
27. WHO.Globaldatabaseonbodymassindex.AccessedFebruary5,2016.Avail‐ ableat:
www.who.int/nutrition/databases/bmi/en/.
28. TanitaAcademy.Understandingyourmeasurements.Availableat:http://tanita.eu/.AccessedMay25,
2018.
29. MatthewsDR,HoskerJP,RudenskiAS,NaylorBA,TreacherDF,TurnerRC.Homeostasismodel
assessment:Insulinresistanceandb‐cellfunctionfromfastingplasmaglucoseandinsulinconcentrations
inman.Diabetologia1985;28:412–9.
30. ReddyY.,MurthyS.,KrishnaD.,PrabhakarM.C.RoleofFreeRadicalsandAntioxidantsinTuberculosis
Patients.IndianJ.Tuberc.2004;51:213–218.
31. VeskoukisA.S.,KyparosA.,PaschalisV.,NikolaidisM.G.SpectrophotometricAssaysforMeasuringRedox
BiomarkersinBlood.Biomarkers.2016;21:208–217.doi:10.3109/1354750X.2015.1126648.
32. JanaszewskaA.,BartoszG.AssayofTotalAntioxidantCapacity:ComparisonofFourMethodsasApplied
toHumanBloodPlasma.Scand.J.Clin.Lab.Invest.2002;62:231–236.doi:10.1080/003655102317475498.
33. KelesM.S.,TaysiS.,SenN.,AksoyH.,AkçayF.EffectofCorticosteroidTherapyonSerumandCSF
MalondialdehydeandAntioxidantProteinsinMultipleSclerosis.Can.J.Neurol.Sci.2001;28:141–143.doi:
10.1017/S0317167100052823.
34. CalderPC,AhluwaliaN,BrounsFetal..Dietaryfactorsandlow‐gradeinflammationinrelationto
overweightandobesity.BrJNutr.2011;106(Suppl.3):S5–78.doi:10.1017/S0007114511005460
35. EmilioRosE.,Martínez‐GonzálezM,EstruchR,Salas‐SalvadóJS,MontserratMFitóM,MartínezJA,
CorellaDMediterraneandietandcardiovascularhealth:TeachingsofthePREDIMEDstudyAdvNutr.
2014May14;5(3):330S‐6S
36. DaiJ,JonesDP,GoldbergJ,ZieglerTR,BostickRM,WilsonPW,ManatungaAK,ShallenbergerL,JonesL,
VaccarinoV.AssociationbetweenadherencetotheMediterraneandietandoxidativestress.AmJClin
Nutr.2008Nov;88(5):1364‐70.
37. FitóM,GuxensM,CorellaDetal.;PREDIMEDStudyInvestigators.EffectofatraditionalMediterranean
dietonlipoproteinoxidation:arandomizedcontrolledtrial.ArchInternMed.2007;167:1195–1203.
doi:10.1001/archinte.167.11.1195[GoogleScholar]
38. TostiV,BertozziB,FontanaL.HealthBenefitsoftheMediterraneanDiet:MetabolicandMolecular
Mechanisms.JGerontolABiolSciMedSci.2018Mar2;73(3):318‐326.
39. CalabreseCM,ValentiniA,CalabreseG.GutMicrobiotaandType1DiabetesMellitus:TheEffectof
MediterraneanDiet.FrontNutr.2021Jan13;7:612773.
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1

11
40. Widmer,R.J.;Flammer,A.J.;Lerman,L.O.;Lerman,A.TheMediterraneandiet,itscomponents,and
cardiovasculardisease.Am.J.Med.2015,128,229–238
41. Hooper,L.V.;Littman,D.R.;Macpherson,A.J.Interactionsbetweenthemicrobiotaandtheimmunesystem.
Science2012,336,1268–1273MD
42. Zhou,N.;Gu,X.;Zhuang,T.;Xu,Y.;Yang,L.;Zhou,M.GutMicrobiota:APivotalHubforPolyphenolsas
Antidepressants.J.Agric.Food.Chem.2020,68,6007–6020
43. EisenbergT,AbdellatifM,SchroederSetal.Cardioprotectionandlifespanextensionbythenatural
polyaminespermidine.NatMed.2016;22:1428–1438.doi:10.1038/nm.4222
44. AlexE.Mohr,CarissaMcEvoy,DorothyD.Sears,PaulJ.Arciero,KarenL.Sweazea,Impactofintermittent
fastingregimensoncirculatingmarkersofoxidativestressinoverweightandobesehumans:Asystematic
reviewofrandomizedcontrolledtrials,AdvancesinRedoxResearch,Volume3,2021,100026
45. McAllister,MJ,Gonzalez,AE,andWaldman,HS.Impactoftimerestrictedfeedingonmarkersof
cardiometabolichealthandoxidativestressinresistance‐trainedfirefighters.JStrengthCondRes36(9):
2515‐2522,2022
46. SuttonEF,BeylR,EarlyKS,CefaluWT,RavussinE,PetersonCM.EarlyTime‐RestrictedFeedingImproves
InsulinSensitivity,BloodPressure,andOxidativeStressEvenwithoutWeightLossinMenwith
Prediabetes.CellMetab.2018Jun5;27(6):1212‐1221.e3.
47. CienfuegosS,GabelK,KalamF,EzpeletaM,WisemanE,PavlouV,LinS,OliveiraML,VaradyKA.Effects
of4‐and6‐hTime‐RestrictedFeedingonWeightandCardiometabolicHealth:ARandomizedControlled
TrialinAdultswithObesity.CellMetab.2020Sep1;32(3):366‐378
48. WimalawansaSJ.VitaminDDeficiency:EffectsonOxidativeStress,Epigenetics,GeneRegulation,and
Aging.Biology(Basel).2019May11;8(2):30
Disclaimer/Publisher’sNote:Thestatements,opinionsanddatacontainedinallpublicationsaresolelythose
oftheindividualauthor(s)andcontributor(s)andnotofMDPIand/ortheeditor(s).MDPIand/ortheeditor(s)
disclaimresponsibilityforanyinjurytopeopleorpropertyresultingfromanyideas,methods,instructionsor
productsreferredtointhecontent.
Preprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 27 August 2024 doi:10.20944/preprints202408.1910.v1