Resting Energy Expenditure, Metabolic and Sex Hormones in Two Phases of the Menstrual and Hormonal Contraceptive Cycles

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY-NC-ND 4.0 h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ Res ing Ene gy Expendi u e, Me abolic and Sex Ho mones in Two Phases o he Mens ual and Ho monal Con acep i e Cycles © 2024 Ame ican College o Spo s Medicine Accep ed e sion (Final d a ) Lö be g, Ida E.; Ka ppinen, Ja i E.; Laa ikainen-Raussi, Vesa; Leh i, Maa i ; Hackney, An hony C.; Ihalainen, Johanna K.; Mikkonen, Ri a S. Lö be g, I. E., Ka ppinen, J. E., Laa ikainen-Raussi, V., Leh i, M., Hackney, A. C., Ihalainen, J. K., & Mikkonen, R. S. (2024). Res ing Ene gy Expendi u e, Me abolic and Sex Ho mones in Two Phases o he Mens ual and Ho monal Con acep i e Cycles. Medicine and Science in Spo s and Exe cise, ahead o P in . h ps://doi.o g/10.1249/mss.0000000000003518 2024 Published ahead o P in Medicine & Science in Spo s & Exe cise® Published ahead o P in con ains a icles in unedi ed manusc ip o m ha ha e been pee e iewed and accep ed o publica ion. This manusc ip will unde go copyedi ing, page composi ion, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed ha could a ec he con en . Copy igh © 2024 Ame ican College o Spo s Medicine Res ing Ene gy Expendi u e, Me abolic and Sex Ho mones in Two Phases o he Mens ual and Ho monal Con acep i e Cycles Ida E. Lö be g1, Ja i E. Ka ppinen1,2, Vesa Laa ikainen-Raussi1, Maa i Leh i1, An hony C. Hackney3, Johanna K. Ihalainen1,4, and Ri a S. Mikkonen5 1Facul y o Spo and Heal h Sciences, Uni e si y o Jy äskylä, Jy äskylä, FINLAND; 2Obesi y Resea ch Uni , Resea ch P og am o Clinical and Molecula Me abolism, Uni e si y o Helsinki, Helsinki, FINLAND; 3 Depa men o Exe cise & Spo Science – Depa men o Nu i ion, Uni e si y o No h Ca olina a Chapel Hill, Chapel Hill, NC; 4Finnish Ins i u e o High Pe o mance Spo KIHU, Jy äskylä, FINLAND; 5Spo s Technology Uni , Facul y Accep ed o Publica ion: 9 July 2024 ACCEPTED Downloaded om h p://jou nals.lww.com/acsm-msse by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hCyw CX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC1y0abggQZXdgGj2MwlZLeI= on 08/09/2024 Res ing Ene gy Expendi u e, Me abolic and Sex Ho mones in Two Phases o he Mens ual and Ho monal Con acep i e Cycles Ida E. Lö be g1, Ja i E. Ka ppinen1,2, Vesa Laa ikainen-Raussi1, Maa i Leh i1, An hony C. Hackney3, Johanna K. Ihalainen1,4, and Ri a S. Mikkonen5 1Facul y o Spo and Heal h Sciences, Uni e si y o Jy äskylä, Jy äskylä, FINLAND; 2Obesi y Resea ch Uni , Resea ch P og am o Clinical and Molecula Me abolism, Uni e si y o Helsinki, Helsinki, FINLAND; 3 Depa men o Exe cise & Spo Science – Depa men o Nu i ion, Uni e si y o No h Ca olina a Chapel Hill, Chapel Hill, NC; 4Finnish Ins i u e o High Pe o mance Spo KIHU, Jy äskylä, FINLAND; 5Spo s Technology Uni , Facul y o Spo and Heal h Sciences, Uni e si y o Jy äskylä, Vuoka i, FINLAND Add ess o co espondence: Ida Lö be g, Facul y o Spo and Heal h Sciences, PO Box 35. FI-40014 Uni e si y o Jy äskylä, Jy äskylä, Finland; E-mail: ida.i.lo be g@jyu. i. Con lic o In e es and Funding Sou ce: The da a p esen ed he e a e pa o a la ge Women’s mens ual cycle and endu ance aining (NaisQs) s udy. The s udy was unded by he Finnish Minis y o Educa ion and Cul u e and Fi s bea Analy ics Expense unding o blood analyses was ecei ed om he Suomen U heilu u kimussää iö. The au ho s decla e no con lic s o in e es , inancial o o he wise, ega ding his s udy. Medicine & Science in Spo s & Exe cise, Publish Ahead o P in DOI: 10.1249/MSS.0000000000003518 ACCEPTED This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion- Non Comme cial-No De i a i es License 4.0 (CCBY-NC-ND), whe e i is pe missible o download and sha e he wo k p o ided i is p ope ly ci ed. The wo k canno be changed in any way o used comme cially wi hou pe mission om he jou nal. ACCEPTED ABSTRACT -lu eal phases o he MC in NoOC-g oup (n=38) o he ac i e and inac i e phases o he COC cycle (COC, n=19). Pa icipan s eco ded hei ood in ake o 3 days a e measu emen s. A seconda y analysis was comple ed o he NoOC-g oup wi hou REE ou lie s (di e ence be ween measu emen s >1.5 × in e qua ile ange, n=4). Resul s: In he NoOC-g oup, lu eal phase REE was 40 kcal highe han ollicula phase REE [95% con idence in e al (CI): -2 kcal/d–82 kcal/d, d=0.20, p=0.061]. Lep in (d=0.35, p<0.001), T3 (d=0.26, p=0.05) and a in ake (d=0.48, p=0.027) we e lowe , and T4 (d=0.21, p=0.041) was highe in he lu eal phase. A e excluding ou lie s, REE was 44 kcal highe in he lu eal phase han in he ollicula phase (95% CI: 12kcal/d–76kcal/d, d=0.22, p=0.007). In he COC-g oup, he mean di e ence in REE was -2 kcal (95% CI-82 kcal/d–79 kcal/d) be ween ac i e and inac i e phases, while T3 was highe in he inac i e phase (d=0.01, p=0.037). Conclusions: REE inc eases only sligh ly om he ollicula o he lu eal phase bu emains unchanged be ween COC phases. Inc eases in T3, lep in, and a in ake du ing he lu eal phase migh echo me abolic luc ua ions ha pa allel emale sex ho mones du ing he MC. ACCEPTED Key Wo ds: FEMALE PHYSIOLOGY, ENERGY INTAKE, HORMONAL CONTRACEPTION, REPRODUCTIVE HORMONES, RESTING METABOLISM ACCEPTED INTRODUCTION The mens ual cycle (MC) is a se ies o inely uned physiological p ocesses caused by luc ua ions in endogenous sex ho mones. These ho monal luc ua ions cause changes no only in he s uc u e and unc ion o he o a ies bu also in luence a ious me abolic pa hways ha egula e ene gy me abolism (1). Res ing ene gy expendi u e (REE) ypically accoun s o 60 o 75% o o al daily ene gy expendi u e and is e lec i e p ima ily o a - ee mass (FFM) and a mass (FM), which a e associa ed wi h unc ional demands o o gans and issues (2). Ne e heless, he possible in luence o sex ho mones on REE emains equi ocal and equi es mo e a en ion o imp o e ou unde s anding o emale physiology (3). The e is some e idence ha REE inc eases in he lu eal phase o he MC compa ed o he ollicula phase. High le els o p oges e one (P4) accompanied by es adiol (E2) du ing he mid-lu eal phase a e p oposed o con ibu e o REE h ough hei he mo egula o y in e ac ions (4,5). In a ecen sys ema ic e iew (3), 47% o 26 s udies epo ed highe REE in he lu eal phase, al hough he a ying quali y o he s udies limi ed he in e p e a ion o he esul s. O pa icula conce n was he inconsis ency o lack o MC phase e i ica ion in included s udies as i has been p oposed ha he MC should be di ided in o ou dis inc phases acco ding o ho monal p o iles when assessing he in luence o sex ho mones and hei a ios on me abolism (3,6). In addi ion o sex ho mones, habi ual ood in ake may inc ease du ing he lu eal phase compa ed o he ollicula phase (7). Reg e ably, no all p e ious esea ch has been able o accoun o habi ual ood in ake, a a iable o signi icance when s udying he ela ionship ACCEPTED be ween he mens ual cycle and me abolism. Conside ing ha ood in ake is closely ela ed o REE (8), MC-dependen pa e ns media ing he e ec s o ood in ake on REE should no be igno ed. Sex ho mones E2 and P4 may in e ac wi h me abolic ho mones ha a e in ol ed in he egula ion o REE and ood in ake. Fo example, hy oid ho mones play an impo an ole in ene gy me abolism by egula ing complex cell unc ions and con ibu ing o he mogenesis and he e o e, s imula ing ene gy expendi u e (9,10). P e ious s udies ha e no , howe e , consis en ly showed ha hy oid ho mone le els luc ua e du ing he MC (11). Adipocy e- de i ed lep in and he gu ho mone gh elin a e conside ed appe i e- egula ing ho mones bu a e also s ongly associa ed wi h ene gy me abolism (12). Lep in is known o be in ol ed in he egula ion o he MC h ough he link be ween ene gy s a us and ep oduc ion and i s inc ease du ing he lu eal phase ha e been consis en ly epo ed (13) while gh elin does no appea o be in luenced by he MC (14). The e is, howe e , e idence ha lep in does no di ec ly in luence he p ocesses unde lying REE (15) whe eas gh elin is shown o be nega i ely associa ed wi h REE independen o body a (16). The syn he ic emale sex ho mones con ained in monophasic combined o al con acep i es (COC) down egula e endogenous E2 and P4 and appea o only i ially a ec o he physiological p ocesses (17). Reg e ably, he in e ac ion o COC wi h REE is no clea , bu a ecen e iew sugges s ha COC use does no a ec ood in ake (18). S udies in es iga ing he e ec s o COC phases on me abolic ho mones a e sca ce, bu cu en ly he e appea s o be ACCEPTED consensus on unchanged hy oid ho mones (19), lep in (20) and gh elin (21) le els du ing he COC cycle. Conside ing ha a woman has an a e age o 450 MCs du ing he li e ime (22), egula luc ua ion in ene gy me abolism can ha e meaning ul e ec s on he heal h. Mo eo e , acco ding o Uni ed Na ions (23), app oxima ely 16% (151 million) o women use COC, which unde lines he impo ance o u he esea ch on he me abolic e ec s o ho monal con acep ion use. As such, he aim o his s udy was o in es iga e he e ec s o endogenous and exogenous emale sex ho mones on REE and me abolic ho mones in na u ally mens ua ing women and women using COC. We hypo hesized ha REE, ene gy in ake, and lep in would be highe in he lu eal phase han in he ollicula phase, while no changes would occu be ween COC cycle phases. MATERIAL AND METHODS Pa icipan s Heal hy un ained women (n = 77, Tie 1: ec ea ionally ac i e, de ined acco ding o he Pa icipan Classi ica ion F amewo k (24)) aged 18-35 yea s wi h a sel - epo ed body mass index (BMI) be ween 19.5 and 35 kg/m2 a ec ui men olun ee ed o pa icipa e in his s udy ia he ad e isemen in social media, spo halls, gyms, public places, and uni e si y mailing lis s. The pa icipan s we e s a i ied in o wo g oups, one no using any ho monal con acep ion wi h a sel - epo ed egula 26–35-day MC o e he p e ious six mon hs (NoOC- g oup, n=58, no mally mens ua ing), and one g oup using monophasic COC (COC-g oup, ACCEPTED S a is ical analysis S a is ical analyses we e conduc ed using SPSS S a is ics 28 (SPSS Inc., Chicago, IL) and igu es we e g aphed wi h G aphPad P ism. Resul s a e epo ed as mean ± SD o as medians wi h i s and hi d qua iles. The e ec size o esul s is exp essed as Cohen’s d (32). The no mali y o he da a was es ed by Shapi o-Wilk. Wi hin-g oup changes we e analyzed by S uden ’s pai ed - es o no mally dis ibu ed da a and Wilcoxon’s signed- ank es o non- no mally dis ibu ed da a. Associa ions be ween a iables o in e es and REE we e es ed by mul iple eg ession analysis using FFM and FM as co a ia es pe ecommenda ions (2). Consis ency wi hin es ing me abolism measu es we e assessed using he in aclass co ela ion coe icien (ICC) wi h a one-way andom-e ec s model (Supplemen al Table 2, Supplemen al Digi al Con en , ICCo REE and RER be ween wo measu emen s in NoOC- and COC- -de e mined le el o s a is ical signi icance was p≤0.05. Inspec ion o he da a e ealed ou pa icipan s in he NoOC g oup as po en ial ou lie s because hei REE change om he ollicula o he lu eal phase exceeded 1.5 × he in e qua ile ange (33). Al hough we ound no physiological o measu emen - ela ed easons o hese obse a ions, ou lie s ha e he po en ial o excessi ely in luence he esul s in pa ame ic es s compa ed wi h o he obse a ions. The e o e, a seconda y analysis was conduc ed, excluding hese ou pa icipan s when using REE as he ou come. RESULTS The physical cha ac e is ics o pa icipan s a e p esen ed in Table 1. Resul s o he NoOC- and COC-g oups a e p esen ed sepa a ely because he g oups a e ho monally di e en , ACCEPTED and he p ima y pu pose o his in es iga ion was o obse e wi hin-g oup changes in wo homogeneous samples. Di e ences be ween ollicula and lu eal phases in he NoOC-g oup Table 2 shows he ho mone concen a ions, es ing me abolism, body composi ion, and die a y measu es du ing he ollicula and lu eal phases. E2 concen a ions we e lowe in he ollicula phase compa ed o he lu eal phase (p<0.001, d=1.83) whe eas all pa icipan s demons a ed P4 alues > 16 nmol/l in he lu eal phase, indica ing expec edly highe P4 concen a ions compa ed o he ollicula phase (p<0.001, d=4.79). Lep in (p<0.001, d=0.35) and T3 (p=0.05, d=0.26) we e lowe in he ollicula phase compa ed o he lu eal phase, while T4 was highe (p=0.042, d=0.21) in he ollicula phase (Figu e 1A–C). In he ull sample, REE da a we e compa ible wi h a -2 kcal/d dec ease o 82 kcal/d inc ease (95% CI) om he ollicula o he lu eal phase (mean di e ence 40 kcal/d, Figu e 2A). Addi ionally, RER emained simila be ween MC phases. Howe e , he seconda y analysis indica ed signi ican ly highe REE in he lu eal phase compa ed o he ollicula phase (mean di e ence 44 kcal/d, 95% CI: 13 kcal/d o 76 kcal/d, p=0.007) (Figu e 2B). We obse ed a s ong ICC o REE in bo h he ull sample and a e excluding ou lie s (p<0.001), whe eas RER showed a weak ICC in bo h cases (p=0.451 and p=0.311, espec i ely) (Supplemen al Table 2, Supplemen al Digi al Con en ). Fa in ake was ound o be highe du ing he lu eal phase compa ed o he ollicula phase (mean di e ence 13 g/d, 95% CI: 2 g/d o 24 g/d, p=0.027) (Figu e 1D). The change in ACCEPTED ene gy in ake was no s a is ically signi ican , bu esul s a o ed highe ene gy in ake du ing he lu eal phase, wi h a mean di e ence o 147 kcal/d (95% CI: -5 kcal/d o 300 kcal/d, p=0.058). Associa ions o ho mones and die a y measu es wi h FFM- and FM-adjus ed REE in he NoOC-g oup The associa ions o ho mone concen a ions and die a y measu es wi h REE adjus ed o FFM and FM a e shown in Table 3. FFM and FM explained 48% and 58% o he a iance o REE in he ollicula and lu eal phases, espec i ely. T3 was posi i ely associa ed wi h REE in bo h phases, inc easing he adjus ed R2 o 51% in he ollicula phase and 69% in he lu eal phase. O he die a y measu es, ene gy in ake (adjus ed R2=55%), ene gy a ailabili y (adjus ed R2=50%), a in ake (adjus ed R2=49%), and ca bohyd a e in ake (adjus ed R2=51%) we e posi i ely associa ed wi h REE in he ollicula phase bu no in he lu eal phase. Seconda y analysis wi hou REE ou lie s indica ed ha he abili y o FFM and FM o explain REE a iabili y imp o ed (adjus ed R2 55% and 64% in he ollicula and lu eal phases, espec i ely). The epo ed associa ions be ween T3 and die a y measu es, and REE emained simila bu we e s onge a e exclusion o ou lie s (Supplemen al Table 3, Supplemen al Digi al Con en , The associa ions o ho mone concen a ions and die a y measu es wi h es ing ene gy expendi u e, adjus ed o FFM and FM, a FOL and LUT phases). T3 inc eased he adjus ed R2 o 64% (β=0.35, p=0.002) in he ollicula phase and 75% (β=0.38, p<0.001) in he lu eal phase. Associa ions be ween REE and ene gy in ake (adjus ed R2=67%, β=0.49, p<0.001), ene gy a ailabili y (adjus ed R2 =60%, β=0.42, p<0.005), a in ake (adjus ed ACCEPTED R2=58%, β=0.37, p<0.010), and ca bohyd a e in ake (adjus ed R2=60%, β=0.40, p<0.005), we e ound in ollicula phase. Di e ences be ween ac i e and inac i e phases in he COC-g oup The ho mone concen a ions, body composi ion, es ing me abolism and die a y measu es in he COC-g oup a e p esen ed in Table 4. T3 (p<0.001, d=0.93) (Figu e 3A) and body mass (p=0.037, d=0.058) we e highe in he inac i e phase compa ed o he ac i e phase, bu T4 and lep in did no change om he ac i e phase o he inac i e phase (Figu e 3B–C). The mean di e ence o REE change be ween phases was -2 kcal (95% CI: -82 kcal/d o 79 kcal/d, p=0.969) (Figu e 3D) and o a in ake -1 g (95% CI: 17 g/d o 15 g/d, p=0.885) (Figu e 3E). The ICC o bo h REE (p=0.005) and RER (p=0.027) was s ong (Supplemen al Table 2, Supplemen al Digi al Con en ). Associa ions o ho mones and die a y measu es wi h FFM- and FM-adjus ed REE in he COC-g oup Table 5 displays associa ions be ween ho mone concen a ions and die a y measu es wi h REE adjus ed o FFM and FM. FFM and FM oge he explained 37% and 66% o he a iance in REE in he ac i e and inac i e phases, espec i ely. Lep in was nega i ely associa ed wi h REE in he inac i e phase, inc easing he adjus ed R2 o 71%, and unAG was nega i ely associa ed wi h REE in he ac i e phase (adjus ed R2=50%). O he ho mones o die a y measu es did no appea o con ibu e o REE along wi h FFM and FM in ei he phase. ACCEPTED DISCUSSION The p esen s udy in es iga ed REE and me abolic ho mones o e one mens ual o COC cycle, o ou knowledge, in he la ges sample o da e. Ou indings sugges ed highe REE du ing he lu eal phase compa ed o he ollicula phase (Figu e 2), while no no able REE di e ences be ween COC cycle phases we e obse ed (Figu e 3D). In he NoOC-g oup, concen a ions o T3, lep in, and a in ake we e highe in he lu eal phase compa ed o he ollicula phase (Figu e 1). Con e sely, in he COC-g oup, only T3 and body mass we e signi ican ly highe du ing he inac i e phase compa ed o he ac i e phase. These indings unde sco e he in ica e ela ionship be ween ho monal luc ua ions and es ing me abolism, emphasizing he necessi y o conside mens ual cycle phase when in e p e ing REE measu emen s. Me abolism and ene gy in ake in he ollicula and lu eal phases Ou esul s sugges ed ha REE was app oxima ely 40 kcal/d highe in he lu eal phase han in he ollicula phase; howe e , he di e ence became s a is ically signi ican only a e excluding ou ou lie s who in luenced he esul s (Figu e 2B). O hese ou lie s, wo had highe REE in he ollicula phase, and he o he wo had highe REE in he lu eal phase. Impo an ly, hei exclusion imp o ed ou abili y o explain REE a ia ion using FFM and FM, jus i ying he alue o ou seconda y analysis. Howe e , despi e in es iga ing he la ges sample o da e, ou s udy may s ill be unde powe ed o d aw p ecise es ima es o he MC e ec on REE. The in aindi idual day- o-day a ia ion in REE has been epo ed o be app oxima ely 5% (34), consis en wi h he es - e es eliabili y obse ed in ou labo a o y. This indica es ha he daily ACCEPTED a ia ion in REE may be e en la ge han he e ec o he MC. The e o e, we ecommend ha ou esul s be in e p e ed in he ligh o p e ious li e a u e. Ne e heless, ou indings a e consis en wi h p e ious sys ema ic e iew and me a-analysis by Ben on e al. (3), who epo ed a sligh ly highe (d=0.33, p<0.001) REE in he lu eal phase compa ed wi h he ollicula phase. Howe e , pa icula ly he inclusion o s udies published in yea 2000 o a e led o e y simila e ec size o ou s (d=0.23, p=0.055). Fu he mo e, whe he he mo e me hodologically compa able p e ious s udies ha e epo ed REE di e ences classi ied as s a is ically signi ican (35) o no (36), he nume ical di e ences be ween ea ly ollicula and lu eal phase end o all in o he same ange as obse ed in ou s udy. Fo ins ance, Day e al. (35) epo ed a 29 kcal highe REE in he lu eal phase, while Ellio e al. (36) obse ed a ia ions anging om 8 kcal o 57 kcal highe REE in he lu eal phase compa ed o he ea ly ollicula phase ac oss wo MCs. The e o e, aking in o conside a ion he o ali y o cu en e idence, he e ec o MC on REE seems p obable. Whe he he e ec has p o ound physiological signi icance emains unce ain. Ou indings sugges ha inc ease o REE could be d i en by T3, which is well known o s imula e ene gy expendi u e (37). In ou s udy, T3 le els showed MC- ela ed luc ua ions ha pa alleled REE and we e associa ed wi h FFM- and FM-adjus ed REE du ing bo h MC phases (Table 3). Howe e , he MC-associa ed T3 esponse has been p e iously epo ed only by La i ie e e al. (38), while o he s udies ha e ound mo e s able T3 le els du ing he MC (21,11,39). This disc epancy could be a ibu ed o he unc ional na u e o hy oid ho mones (40), coupled wi h me hodological di e ences such as a ia ions in sample ACCEPTED iming and assay echniques ac oss s udies. We also no ed highe lep in concen a ions in he lu eal phase (Figu e 1C), consis en wi h p e ious esea ch (13,21,41). Howe e , lep in le els we e no associa ed wi h REE, which ag ees wi h p e ious esea ch (15). O o he po en ial me abolic ho mones, we ound s able le els o UnAG and AG h oughou he MC. Mo eo e , hei le els we e no associa ed wi h REE, indica ing ha hese ho mones a e una ec ed by MC and a e unlikely o explain MC-associa ed changes in REE. Ne e heless, u he in es iga ion is equi ed o elucida e he complex in e play be ween emale sex ho mones, o he me abolic ho mones, and ene gy expendi u e. Conside ing ene gy in ake as a pa o he ene gy balance equa ion, we obse ed highe a in ake du ing he lu eal phase compa ed o he ollicula phase (Figu e 1D), which is in line wi h ew p e ious s udies (42, 43). Fo ene gy in ake, ou da a indica ed i o emain cons an o inc ease om he ollicula o he lu eal phase, o which he la e ag ees wi h he widely held iew ha ene gy in ake is highe du ing he lu eal phase compa ed o he la e ollicula phase (7,44–46). The hypo hesized physiological eason o hese a ia ions is ha E2 may inhibi appe i e in he la e ollicula phase (47) ia a complex p ocess in ol ing hypo halamic E2 neu ons and hei egula o y ole in ene gy homeos asis (48). Con e sely, P4 is sugges ed o s imula e appe i e in he p esence o E2 du ing he lu eal phase (47). These changes could also be a esponse o he possible inc ease in REE o main ain ene gy balance (49). Howe e , an inc ease in ene gy in ake du ing he lu eal phase has no consis en ly been epo ed (21,50). I should be emphasized ha he con ol o ood in ake is in luenced by psychological and social ac o s as well as physiological mechanisms (51), which unde lines he unce ain y o e alua ing ene gy in ake da a. ACCEPTED In his s udy, all die a y measu es, excep p o ein in ake, con ibu ed o he FFM- and FM-adjus ed REE in he ollicula phase (Table 3). In he long- e m, ene gy in ake na u ally e lec s body size, which explains he ela ionship be ween REE and ene gy in ake (8,52). Die p io o REE measu emen is known o in luence he esul s h ough he inc eased he mic e ec o ood (26). Howe e , he ein die a y in ake was eco ded o 3 days a e he REE measu emen o p ac ical easons. Ne e heless, he s able RER obse ed be ween he ollicula and lu eal phase indica es a lack o d ama ic changes in ene gy balance and subs a e me abolism p eceding he measu emen (53), which is also suppo ed by he lack o signi ican changes in ene gy in ake acco ding o he ood dia ies in his s udy. Addi ionally, REE measu emen was conduc ed in a as ed s a e o mi iga e he mic e ec o ood. The unde lying eason o he associa ions obse ed only du ing he ollicula phase, bu no lu eal phase, is unknown. Howe e , i can be specula ed ha he ene gy in ake eplaced some o he mass- ela ed aspec s o REE in he ollicula phase. This specula ion is suppo ed by ou inding ha FFM and FM explained a smalle p opo ion o REE a iabili y in he ollicula phase han in he lu eal phase (48% s. 58%). Fu he mo e, adding ene gy in ake o he model inc eased he explana o y pe cen age om 48% o 59% in he ollicula phase, simila o he explana o y pe cen age o FFM and FM (58%) in he lu eal phase. This obse a ion implies ha ene gy in ake migh play a mo e signi ican ole in REE a iabili y in he ollicula phase, p o iding insigh in o he complex na u e o ene gy me abolism in women and indica ing he need o u he in es iga ion. ACCEPTED Me abolism and ene gy in ake in he ac i e and inac i e phases This s udy may be he i s o examine REE di e ences be ween he ac i e and inac i e phase o he COC cycle while also epo ing ho monal concen a ions. In he p esen s udy, bo h endogenous E2 and P4 emained s able du ing he COC cycle as expec ed. Ou esul s indica e no e idence o a di e ence in REE be ween COC phases (Figu e 3), which ag ees wi h p e ious s udies (54,55). In he p esen s udy, only T3 was highe du ing he inac i e phase o he COC cycle (Figu e 3A), which con adic s wi h p e ious s udies ha epo ed s able hy oid ho mone le els h oughou he COC cycle (19,21). This unexpec ed inding indica es ha u he in es iga ion o po en ial ac o s con ibu ing his a ia ion in hy oid ho mone le els du ing he COC cycle migh be equi ed. Howe e , i is no ewo hy ha highe T3, T4, and TSH le els ha e been epo ed in COC-use s compa ed o non-use s (19) as well as a commencemen o COC use (56,57). This di e ence is hough o be ela ed o he inc ease o hy oxine-binding globulin caused by COCs (56). Ne e heless, highe T3 in he inac i e phase o he COC cycle migh be a ibu ed o indi idual a ia ions in hy oid me abolism o o he unexplained ac o s. - and FM-adjus ed REE was no associa ed wi h T3 a ei he phase, bu lep in was in e sely co ela ed wi h REE in he inac i e phase while unAG had a nega i e co ela ion wi h REE in he ac i e phase (Table 5). The inabili y o T3 o explain REE a ia ion in he COC- g oup aises ques ions abou he in luence o exogenous emale sex ho mones on hy oid ho mone me abolism. Fu he mo e, he unde lying physiological mechanisms d i ing hese obse ed nega i e ela ionships be ween REE and lep in, as well as REE and UnAG, emain ACCEPTED inconclusi e and wa an u he in es iga ion. Unde s anding hese associa ions may se e as a means o op imize me abolic heal h o women using COCs. Gi en ha only ew s udies ha e add essed wi hin-cycle changes in body composi ion and die a y in ake among COC use s, ou s udy is a aluable con ibu ion o his a ea o esea ch. We obse ed highe body mass du ing he inac i e phase, while FM, FFM, and die a y in ake emained unchanged. Some p e ious s udies ha e epo ed ha body mass and body composi ion do no change du ing he COC cycle (58,59). Howe e , ou esul s align wi h Ihalainen e al. (21) who ound highe body mass du ing he inac i e phase, sugges ing ha COC use migh ha e a ole in body weigh luc ua ions. Indeed, E2 inc eases plasma olume, whe eas P4 is also known o in luence sodium and wa e egula ion (60). S udies examining die a y in ake ac oss he COC phases ha e consis en ly epo ed no changes (21,54,61), which is suppo ed by ou indings. I can be specula ed ha in addi ion o inhibi ing luc ua ions in emale sex ho mones, he use o COC migh also s abilize he assumed cyclic luc ua ions in ene gy in ake. S eng hs and limi a ions The majo s eng h o his s udy was he ca e ul e i ica ion o he MC phases. In he NoOC-g oup, all pa icipan s had ho monally indica ed o ula ions combined wi h P4 concen a ions o e 16 nmol/l du ing he lu eal phase, which ensu ed he desi ed con ex o in es iga ing me abolism in wo ho monally di e en en i onmen s. Fu he mo e, we c i ically assessed he sample included in he analysis, as well as con olled pa icipan s’ p epa a ion and moni o ed die a y in ake. ACCEPTED 27. Wei JB. New me hods o calcula ing me abolic a e wi h special e e ence o p o ein me abolism. J Physiol. 1949;109(1-2):1-9. 28. Walke S, Sahinaho UM, Vekki S. e al. Two-week s ep- educ ion has limi ed nega i e e ec s on physical unc ion and me abolic heal h in olde adul s. Eu J Appl Physiol. 2024;124(7):2019-33. 29. Hopkins WG. Measu es o eliabili y in spo s medicine and science. Spo s Med. 2000;30(1):1–15. 30. Robe son RJ. Pe cei ed Exe ion o P ac i ione s: Ra ing E o Wi h he OMNI Pic u e Sys em. Champaign, IL: Human Kine ics; 2004. 31. Ainswo h BE, Haskell WL, He mann SD, e al. Compendium o Physical Ac i i ies: a second upda e o codes and MET alues. Med Sci Spo s Exe c. 2011;43(8):1575–81. 32. Cohen J. S a is ical Powe Analysis o he Beha io al Sciences. 2nd ed. Hillsdale (NJ): Law ence E lbaum Associa es; 1988. 567 p. 33. Wal ish S. A e iew o s a is ical ou lie me hods. Pha m Technol. 2006;30(11):82–86. 34. Bade N, Bosy-Wes phal A, Dilba B, Mülle MJ. In a- and in e indi idual a iabili y o es ing ene gy expendi u e in heal hy male subjec s -- biological and me hodological a iabili y o es ing ene gy expendi u e. B J Nu . 2005;94(5):843-9. 35. Day DS, Goz supp ession educes es ing ene gy expendi u e and β-ad ene gic suppo o es ing ene gy expendi u e. J Clin Endoc inol Me ab. 2005;90(6): 3312-7. 36. Ellio SA, Ng J, Leow MK, e al. The in luence o he mens ual cycle on ene gy balance and as e p e e ence in Asian Chinese women. Eu J Nu . 2015;54(8):1323–32. ACCEPTED 37. Ya uz S, Salgado Nunez DPS, Celi FS. Thy oid ho mone ac ion and ene gy expendi u e. J Endoc Soc. 2019;3(7):1345-56. 38. La i ie e F, Moussalli R, Ga el D. Inc eased leucine lux and leucine oxida ion du ing he lu eal phase o he mens ual cycle in women. Am J Physiol Endoc inol Me ab. 1994;267(3):E422–8. 39. Bai X, Li J, Zhou L, Li X. In luence o he mens ual cycle on nonlinea p ope ies o hea a e a iabili y in young women. Am J Physiol Hea Ci c Physiol. 2009;297(2):H765-74. 40. Jacobson MH, Howa ds PP, Da ow LA, e al. Thy oid ho mones and mens ual cycle unc ion in a longi udinal coho o p emenopausal women. Paedia Pe ina Epidemiol. 2018;32(3):225–34. 41. Ah ens K, Mum o d SL, Schliep KC, e al. Se um lep in le els and ep oduc i e unc ion du ing he mens ual cycle. Am J Obs e Gynecol. 2014;210(3):248e1–9. 42. Chappell S, Hackney AC. Associa ions be ween mens ual cycle phase, physical ac i i y le el and die a y mac onu ien in ake. Biol Spo . 1997;14(4):251-8. 43. McNeil J, Came on JD, Finlayson G, Blundell JE, Douce E. G ea e o e all ol ac o y pe o mance, explici wan ing o high a oods and lipid in ake du ing he mid-lu eal phase o he mens ual cycle. Physiol Beha . 2013;112–113:84–89. 44. B ennan IM, Fel in KL, Nai NS, e al. E ec s o he phases o he mens ual cycle on gas ic emp ying, glycemia, plasma GLP-1 and insulin, and ene gy in ake in heal hy lean women. Am J Physiol Gas oin es Li e Physiol. 2009;297(3):G602–10. 45. Gil YRC, Fagundes RLM, San os E, e al. Rela ion o mens ual cycle and alimen a y consump ion o women. Clin Nu ESPEN. 2009;4:e257–60. ACCEPTED 46. Chung SC, Bond EF, Ja e ME. Food in ake changes ac oss he mens ual cycle in Taiwanese women. Biol Res Nu s. 2010;12(1):37-46. 47. Hi schbe g AL. Sex ho mones, appe i e and ea ing beha iou in women. Ma u i as. 2012;71(3):248–56. 48. F ank A, B own LM, Clegg DJ. The ole o hypo halamic es ogen ecep o s in me abolic egula ion. F on Neu oendoc inol. 2014;35(4):550–7. 49. Webb P. 24-hou ene gy expendi u e and he mens ual cycle. Am J Clin Nu . 1986;44(5):614–9. 50. Go czyca AM, Sjaa da LA, Mi chell EM, e al. Changes in mac onu ien , mic onu ien , and ood g oup in akes h oughou he mens ual cycle in heal hy, p emenopausal women. Eu J Nu . 2016;55(3):1181-8. 51. Hopkins M, Beaulieu K, Gibbons C, e al. The Con ol o Food In ake in Humans. In: Feingold KR, Anawal B, Blackman MR, e al., edi o s. Sou h Da mou h (MA): MDTex .com, Inc.; 2000-. 52. Hopkins M, Finlayson G, Dua e C, e al. Modelling he associa ions be ween a - ee mass, es ing me abolic a e and ene gy in ake in he con ex o o al ene gy balance. In J Obes. 2016;40(2):312–8. 53. Go is AH, Wes e e p KR. Pos abso p i e espi a o y quo ien and ood quo ien -an analysis in lean and obese men and women. Eu J Clin Nu . 2000;54(7):546-50. 54. Eck LH, Benne AG, Egan BM, e al. Di e ences in mac onu ien selec ions in use s and nonuse s o an o al con acep i e. Am J Clin Nu . 1997;65(2):419-24. ACCEPTED 55. Duhi a MR, Schu z Y, Mon ani JP, Dulloo AG, Miles-Chan JL. O al con acep i e pill al e s acu e die a y p o ein-induced he mogenesis in young women. Sp ing). 2017;25(9):1482-5. 56. Wieg a z I, Ku sche a E, Lee JH, e al. E ec o ou o al con acep i es on hy oid ho mones, ad enal and blood p essu e pa ame e s. Con acep ion. 2003;67(5):361–6 57. Sänge N, S ahlbe g S, Man hey T, e al. E ec s o an o al con acep i e con aining 30 mcg e hinyl es adiol and 2 mg dienoges on hy oid ho mones and and ogen pa ame e s: con en ional s. ex ended-cycle use. Con acep ion. 2008;77(6):420-5. 58. Vaiksaa S, Jü imäe J, Mäes u J, e al. No e ec o mens ual cycle phase on uel oxida ion du ing exe cise in owe s. Eu J Appl Physiol. 2011;111(6):1027-34. 59. Rael B, Al a o-Magallanes VM, Rome o-Pa a N, e al. Mens ual cycle phases in luence on ca dio espi a o y esponse o exe cise in endu ance- ained emales. In J En i on Res Public Heal h. 2021;18(3):860. 60. S achen eld NS. Sex ho mone e ec s on body luid egula ion. Exe c Spo Sci Re . 2008;36(3):152–9. 61. Tucci SA, Mu phy LE, Boyland EJ, Dye L, Hal o d JC. O al con acep i e e ec s on ood choice du ing he ollicula and lu eal phases o he mens ual cycle. A labo a o y based s udy. Appe i e. 2010;55(3):388-92. 62. Bu ows M, Pe e s CE. The in luence o o al con acep i es on a hle ic pe o mance in emale a hle es. Spo s Med. 2007;37(7):557–74. 63. Miles-Chan JL, Dulloo AG, Schu z Y. Fas ing subs a e oxida ion a es assessed by indi ec calo ime y: is p io die a y mac onu ien le el and composi ion a con ounde ? In J Obes (Lond). 2015;39(7):1114-7. ACCEPTED FIGURE LEGENDS Figu e 1. Changes in T3 (A), n=38, T4 (B), n=38, lep in (C), n=38, and a in ake (D), n=29, a ollicula phase (FOL) and lu eal phase (LUT). Da a a e p esen ed as means wi h s anda d de ia ion and indi idual da a poin s. NoOC. *p≤0.05, ***p<0.001 Figu e 2. Changes in REE in he ull sample (A), n=38, and a e he seconda y analysis (B), n=34, a ollicula phase (FOL) and lu eal phase (LUT). Da a a e p esen ed as means wi h s anda d de ia ion and indi idual da a poin s. NoOC. **p≤0.01 Figu e 3. Changes in T3 (A), n=19, T4 (B), n=19, lep in (C), n=19, REE (D), n=19, and a in ake (E), n=16, a ac i e phase (ACT) and inac i e phase (INACT). Da a a e p esen ed as means wi h s anda d de ia ion and indi idual da a poin s. COC. ***p<0.01 ACCEPTED SUPPLEMENTAL DIGITAL CONTENT ACCEPTED ACCEPTED ACCEPTED ACCEPTED Table 1. Mean ± SD physical cha ac e is ics o he NoOC- (n=38) and COC-g oup (n=19). NoOC-g oup COC-g oup Age (y ) 29.8 ± 3.9 26.1±4.0 Heigh (m) 1.66 ± 0.06 1.69 ± 0.05 Body mass (kg) 68.3 ± 10.9 68.5 ± 6.7 BMI (kg·m−2) 24.9 ± 4.0 24.1 ± 2.1 Body a (%) 28.3 ± 8.3 27.7 ± 6.3 Leng h o MC o COC cycle (days) 27 ± 2 21–24 ac i e + 4–7 inac i e NoOC: na u ally mens ua ing; COC: combined o al con acep i e; BMI: body mass index; COC: combined o al con acep i e. Signi ican di e ences bolded (p≤0.05). ACCEPTED Supplemen al Digi al Con en 1 Supplemen al Table 1. Con en o COC pills and b and names used by pa icipan s in he COC-g oup. B and name P oges ogen componen (gene a ion) N Ges inyl E hinyles adiol 20 µg Ges oden 75 µg (3 d) 3 Ges inyl E hinyles adiol 30 µg Ges oden 75 µg (3 d) 2 Mic ogynon E hinyles adiol 0,15 mg Le ono ges el 30 µg (2nd) 1 Tasmine a/Yasmin E hinyles adiol 0,03 mg D ospi enone 3 mg (4 h) 2 Daisynelle E hinyles adiol 20 µg Desoges el 150 µg (3 d) 1 Dieno e e E hinyles adiol 0,03 mg Dienoges 2 mg (4 h) 5 Yasminelle/Dizminelle/S e aminelle E hinyles adiol 0,02 mg D ospi enone 3 mg (4 h) 3 Me cilon E hinyles adiol 20 µg Desoges el 150 µg (3 d) 1 Le esia E hinyles adiol 20 µg Le ono ges el 100 µg (2nd) 1 COC: combined o al con acep i e ACCEPTED Supplemen al Figu e 1. Supplemen al Figu e 1. Flowcha o pa icipan en ollmen h ough he s udy. FOL: ollicula phase; ACT: ac i e phase; LUT: lu eal phase; INACT: inac i e phase; P4: p oges e one; MC: mens ual cycle; TSH: hy oid-s imula ing ho mone. ACCEPTED Supplemen al Table 2. In aclass co ela ion coe icien s (ICC) o REE and RER be ween wo measu emen s in NoOC- and COC-g oups. Resul s o he seconda y analysis in i alics. Va iable G oup ICC (95%CI) P- alue REE NoOC 0.88 (0.76–0.94) 0.94 (0.87–0.97) <0.001*** <0.001*** COC 0.71 (0.26–0.89) 0.005* RER NoOC 0.04 (-0.84–0.50) 0.16 (-0.68–0.58) 0.451 0.311 COC 0.60 (-0.01–0.85) 0.027* REE: es ing ene gy expendi u e; RER: espi a o y exchange a io. *p≤0.05 ***p<0.001. S a is ically signi ican p- alues a e bolded. ACCEPTED Supplemen al Table 3. The associa ions o ho mone concen a ions and die a y measu es wi h es ing ene gy expendi u e, adjus ed o a - ee mass (FFM) and a mass (FM), a ollicula (FOL) and lu eal (LUT) phases. Resul s o he seconda y analysis. NoOC. FOL LUT B 95% CI β P- alue n B 95% CI β P- alue n Ho mones E2 (nmol/l) 0.04 -0.21 o 0.28 0.04 0.757 34 -0.07 -0.35 o 0.21 -0.07 0.608 34 P4 (nmol/l) 22.12 -62.55 o 106.78 0.07 0.598 34 0.04 -0.21 o 0.28 0.04 0.757 34 T3 (pmol/l) 81.99 31.58 o 132.41 0.35 0.002 34 95.56 50.16 o 140.96 0.38 <0.001 34 T4 (pmol/l) 22.31 -12.52 o 57.14 0.17 0.201 34 1.88 -23.78 o 27.53 0.018 0.882 34 TSH (mIU/l) -14.98 -74.38 o 44.43 -0.06 0.610 34 39.22 -7.11 o 85.56 0.18 0.094 34 Lep in (ng/ml) 3.32 -4.84 o 11.47 0.19 0.413 34 2.42 -4.31 o 9.15 0.18 0.468 34 unAG (pg/ml) 0.12 -0.16 o 0.41 0.11 0.387 33 0.14 -0.07 o 0.36 0.15 0.182 34 AG (pg/ml) 0.11 -0.21 o 0.42 0.09 0.494 33 0.08 -0.15 o 0.31 0.08 0.486 34 Die a y measu es EI (kcal·day−1) 0.21 0.10 o 0.32 0.49 <0.001 29 0.08 -0.03 o 0.18 0.22 0.133 27 EA (kcal·day−1) 0.21 0.07 o 0.35 0.42 0.005 27 0.08 -0.03 o 0.19 0.22 0.132 26 FAT (g·day−1) 2.52 0.65 o 4.39 0.37 0.010 29 1.89 -0.12 o 3.90 0.29 0.065 27 CHO (g·day−1) 1.30 0.43 o 2.17 0.40 0.005 29 0.34 -0.46 o 1.14 0.12 0.394 27 PROT (g·day−1) 0.53 -1.43 o 2.50 0.08 0.582 29 0.41 -1.64 o 2.46 0.07 0.681 27 P- alues <0.05 in bold. B: uns anda dized coe icien ; CI: con idence in e al; β: s anda dized coe icien ; E2: es adiol; P4: p oges e one; T3: iiodo hy onine; T4: hy oxine; TSH: hy oid-s imula ing ho mone; unAG; unacyla ed gh elin; AG: acyla ed gh elin; EI: ene gy in ake, EA: ene gy a ailabili y; FAT: a in ake; CHO: ca bohyd a e in ake; PROT: p o ein in ake. ACCEPTED