Impact of the Method Used to Select Gas Exchange Data for Estimating the Resting Metabolic Rate, as Supplied by Breath-by-Breath Metabolic Carts

nu ien s A icle Impac o he Me hod Used o Selec Gas Exchange Da a o Es ima ing he Res ing Me abolic Ra e, as Supplied by B ea h-by-B ea h Me abolic Ca s Juan M.A. Alcan a a 1,* , Guille mo Sanchez-Delgado 1,2, F ancisco J. Ama o-Gahe e 1,3 , Jose E. Galgani 4and Jona an R. Ruiz 1 1 PROFITH “PROmo ing FITness and Heal h Th ough Physical Ac i i y” Resea ch G oup, Spo and Heal h Uni e si y Resea ch Ins i u e (iMUDS), Depa men o Physical and Spo s Educa ion, Facul y o Spo Sciences , Uni e si y o G anada, 18011 G anada, Spain; gsanchezdelgado@ug .es (G.S.-D.); ama o @ug .es (F.J.A.-G.); uizj@ug .es (J.R.R.) 2Penning on Biomedical Resea ch Cen e , Ba on Rouge, LA 70808, USA 3EFFECTS-262 Resea ch G oup, Depa men o Physiology, School o Medicine, Uni e si y o G anada, 18011 G anada, Spain 4Depa amen o de Ciencias de la Salud, Ca e a de Nu ición y Die é ica, Facul ad de Medicina, Pon i icia Uni e sidad Ca ólica de Chile, San iago, Chile; Depa amen o de Nu ición, Diabe es y Me abolismo, Facul ad de Medicina, Uni e sidad Pon i icia Ca ólica de Chile, 8330023 San iago, Chile.; [email p o ec ed] *Co espondence: alcan a ajma@ug .es; Tel.: +34-958-244-353 Recei ed: 23 Decembe 2019; Accep ed: 10 Feb ua y 2020; Published: 14 Feb ua y 2020   Abs ac : The me hod used o selec ep esen a i e gas exchange da a om la ge da ase s in luences he es ing me abolic a e (RMR) e u ned. This s udy de e mines which o h ee me hods yields he lowes RMR (as ecommended o use in human ene gy balance s udies), and in which me hod he g ea es a iance in RMR is explained by classical de e minan s o his a iable. A o al o 107 young and 74 middle-aged adul s unde wen a 30 min RMR examina ion using a b ea h-by-b ea h me abolic ca . Th ee gas exchange da a selec ion me hods we e used: (i) s eady s a e (SS ) o 3, 4, 5, o 10 min, (ii) a p e-de ined ime in e al (TI), i.e., 6–10, 11–15, 16–20, 21–25, 26–30, 6–25, o 6–30 min, and (iii) “ il e ing”, se ing h esholds depending on he mean RMR alue ob ained. In bo h coho s, he RMRs yielded by he SS and il e ing me hods we e signi ican ly lowe (p<0.021) han hose yielded by he TI me hod. No di e ences in RMR we e seen unde he di e en condi ions o he SS me hod, o o he il e ing me hod. No di e ences we e seen be ween he me hods in e ms o he a iance in RMR explained by i s classical de e minan s. In conclusion, he SS and il e ing me hods e u n he lowes RMRs and in a-measu emen coe icien s o a ia ion when using b ea h-by-b ea h me abolic ca s. Keywo ds: es ing ene gy expendi u e; CCM exp ess; CPX Ul ima Ca diO2; indi ec calo ime y; mac onu ien oxida ion 1. In oduc ion The es ing me abolic a e (RMR) is he lowes ene gy expendi u e o a pe son who is awake [ 1 ], a e a leas 12 h o as ing, being in physical es , and in a s a e o men al elaxa ion in an ambien en i onmen al empe a u e; i accoun s o some 60–70% o he o al daily ene gy expendi u e [ 2 ]. The assessmen o RMR is impo an when s udying human ene gy balance, bo h in clinical and esea ch se ings [ 2 , 3 ]. Indi ec calo ime y is he e e ence me hod o assessing RMR [ 2 , 4 – 6 ], which is es ima ed om he consump ion o oxygen (VO 2 ) and he p oduc ion o ca bon dioxide (VCO 2 ) [ 6 ]. The measu emen o VO 2 and VCO 2 , oge he wi h u ina y ni ogen exc e ion, also allows o he Nu ien s 2020,12, 487; doi:10.3390/nu12020487 www.mdpi.com/jou nal/nu ien s Nu ien s 2020,12, 487 2 o 14 es ima ion o he nu ien (ca bohyd a e and a ) oxida ion a e [ 7 ]. Indeed, he VCO 2 /VO 2 a io, i.e., he espi a o y quo ien (RQ), is an indica o o he ela i e p edominance o a (FATOx) and ca bohyd a e (CHOOx) oxida ion. The assessmen o RMR using indi ec calo ime y is no mally pe o med o e a 10–30 min pe iod. I is widely assumed ha he i s 5 min o da a eco ded should be disca ded [ 8 , 9 ]. A sho (e.g., 5 min) s eady espi a o y s a e pe iod, i.e., a pe iod in which he indi ec calo ime y eco d is ma kedly s able, hen has o be selec ed om he emaining da ase o es ima ing he RMR [ 6 , 7 , 10 ]. The assump ion ha s eady s a e (SS ) me hods o gas exchange da a selec ion p o ide a be e es ima e o RMR han he o he me hods a ailable a ose om s udies pe o med in en ila ed pa ien s [ 10 ]. Howe e , he e is no s ong e idence ha he same can be assumed in heal hy, non- en ila ed subjec s—and indeed di e en me hods ha e been used. These me hods can be g ouped in o h ee ca ego ies: (i) he selec ion o an SS (de ined as ha p o iding a coe icien o a iance [CV] o <10% o VO 2 , VCO 2 , and minu e en ila ion [VE], and o <5% o RQ [ 11 ]), (ii) he selec ion o a p e-de ined ime in e al (TI), wi hou aking he s abili y o he esul s ob ained in o conside a ion [ 11 ], and (iii) “ il e ing”, in which da a abo e o below a gi en RMR h eshold a e disca ded. Bo h he SS and TI me hods can be used unde di e en ime condi ions [ 1 ]. Un o una ely, he use o di e en me hods o gas exchange da a selec ion could esul in di e en es ima es o RMR and nu ien oxida ion a es being made [ 1 , 11 , 12 ]. Fo ins ance, in a s udy in ol ing heal hy subjec s, I ing e al. [ 1 ] epo ed RMR es ima es made by he SS and TI me hods o di e by some −101 o +121 kcal/day. Ce ainly, SS -based RMR es ima es a e usually lowe han hose p o ided by he TI me hod [ 1 , 11 ]. Gi en he abo e de ini ion o RMR [ 1 ], i has been p oposed ha he lowes es ima es ob ained by he SS me hod should be deemed mo e accu a e han hose p o ided by TI. Howe e , he unde -es ima ion o he homeos a ic RMR canno be uled ou in he SS me hod, no has any s udy checked whe he he il e ing me hods o da a selec ion p o ide lowe RMR es ima es han ei he SS o TI. The p esen wo k examines whe he he SS , TI, o il e ing me hod yields he lowes RMR alue in heal hy, non- en ila ed subjec s, and de e mines in which me hod he g ea es a iance in RMR is explained by he classical de e minan s o his a iable (i.e., body weigh , body composi ion, and sex) [13]. 2. Ma e ials and Me hods 2.1. S udy Subjec s The pa icipan s o his e ospec i e s udy we e 107 young adul s (72 women) en olled in he ACTIBATE s udy [ 14 ] and 74 middle-aged adul s (39 women) en olled in he FIT-AGEING s udy [ 15 ]. De ailed in o ma ion abou he me hodology o he a o emen ioned s udies can be ound elsewhe e [ 14 , 15 ]. B ie ly, he inclusion c i e ia we e: (i) being physically inac i e (<20 min o mode a e– igo ous physical ac i i y on <3 days/week), (ii) ha ing a s able body weigh (change <3 kg o e he las 3 mon hs [ACTIBATE] o <5 kg o e he las 5 mon hs [FIT-AGEING]), (iii) no being en olled in a weigh loss p og am, (i ) no being a smoke , ( ) no su e ing om an acu e o ch onic illness, and ( i) no being p egnan . The ACTIBATE s udy p o ocol was app o ed by he Commi ee o Resea ch In ol ing Human Subjec s a he Uni e si y o G anada (Re e ence #924) and he Se icio Andaluz de Salud (Cen o de G anada, CEI-G anada), while he FIT-AGEING was app o ed by he Human Resea ch E hics Commi ee o he Jun a de Andaluc í a(0838-N-2017). Bo h s udies we e pe o med in acco dance wi h he Decla a ion o Helsinki (2013 e ision) and egis e ed on he clinical ials.go pla o m (IDs: NCT02365129 o he ACTIBATE s udy and NCT03334357 o he FIT-AGEING s udy). O al and w i en in o med consen was ob ained om all he subjec s be o e hei en olmen . 2.2. P ocedu es In bo h he abo e s udies, subjec s unde wen a 30 min indi ec calo ime y assessmen o RMR a es , ea ly in he mo ning, ollowing an o e nigh as . All subjec s we e ins uc ed o e ain om Nu ien s 2020,12, 487 3 o 14 mode a e (24 h) and igo ous physical ac i i y (48 h) be o e he es day. On he p e ious e ening, all subjec s consumed a s anda dized meal o an egg omele , boiled ice, and oma o sauce (ad libi um amoun s). They we e also ins uc ed o a oid physical ac i i y a e hey woke up, and o come o he esea ch cen e by ca o bus ea ly in he mo ning, ha ing had no b eak as (ensu ing a ~ 12 h as ) . Upon a i al, and a e con i ming hei compliance wi h hese abo e condi ions, body weigh and heigh we e measu ed using a Seca model 799 elec onic column scale (seca GmbH & Co. KG, Hambu g, Ge many) wi h subjec s ba e oo and wea ing ligh clo hing. The ea e , he subjec s laid on a bed in he supine posi ion o 20–30 min. Gas exchange da a we e hen eco ded by indi ec calo ime y o 30 min in a quie oom wi h dim ligh ing, con olled a 22–24 ◦ C and 35–45% ela i e humidi y [ 8 ]. Du ing his ime he subjec s we e co e ed wi h a bed shee and ins uc ed o emain silen , s ay awake, a oid idge ing, and o b ea he no mally. 2.3. Gas Exchange Assessmen s Gas exchange was eco ded using ei he a CCM Exp ess o a CPX Ul ima Ca diO2 ( wo di e en de ices we e used only in he middle-aged adul s coho ) b ea h-by-b ea h me abolic ca (Medical G aphics Co p, S . Paul, MN, USA). Bo h ins umen s equi e he use o a ace mask equipped wi h a Di ec connec ™ low senso (Medical G aphics Co p, S . Paul, MN, USA). Bo h de e mine VCO 2 using a non-dispe si e in a ed analyze , and bo h de e mine VO 2 using a gal anic uel cell [ 16 ]. The low a e was calib a ed using a 3 L sy inge a he beginning o e e y es . The gas analyze s we e calib a ed be o e each measu emen using s anda d gases acco ding o he manu ac u e s’ ins uc ions [16]. 2.4. Me hods o Gas Exchange Da a Selec ion The collec ed gas exchange da a we e p ocessed using MGCDiagnos ic ® B eeze Sui e 8.1.0.54 SP7 so wa e (Medical G aphics Co p., S . Paul, MN, USA) o yield a da a poin o each a iable o e e y minu e (i.e., he means o all en ila ion da a (pe minu e en ila ion da a—pMVD) o each pa icula minu e). The i s 5 min o da a we e disca ded [ 8 , 9 ]; he emaining 25 min pe iod da ase was p ocessed using h ee di e en me hods o selec ep esen a i e gas exchange da a o de e mining he RMR and nu ien oxida ion a e. 2.4.1. Time In e al Me hod Sho TIs o 6–10 min, 11–15 min, 16–20 min, 21–25 min, and 26–30 min, and long TIs o 6–25 min and 6–30 min we e es ablished [ 11 ], and he means o he pMVD alues o all a iables a ailable o hese ime pe iods calcula ed. These p ocessed da a we e used o calcula e he RMR and nu ien oxida ion a e (see below o de ails). 2.4.2. S eady-S a e Time Me hod The CVs o VO 2 , VCO 2 , VE, and RQ we e calcula ed o e e y pe iod o 3, 4, 5, and 10 min (e.g., o he 3 min SS we p ocessed all he 25 min pe iod da ase s and we examined he 6 h o 8 h min pe iod, he 7 h o 9 h pe iod, e c.) and he mean CVs o each a iable calcula ed o each ime pe iod. The pe iods selec ed o he inal analyses we e hose wi h he lowes mean CV o each (e.g., om he 3 min SS examined pe iods, we selec ed he 7 h o 9 h) [ 11 ]. The means o he a ailable pMVD alues o hese ime pe iods we e hen calcula ed. These p ocessed da a we e used o calcula e he RMR and nu ien oxida ion a e (see below o de ails). 2.4.3. Fil e ing Me hod The pMVD alues o VO 2 and VCO 2 o he en i e 25 min da a collec ion pe iod—i.e., wi h no di ision in o SS o TI pe iods—we e used o calcula e he mean 25 min RMR (see below o de ails). Fu he mo e, pMVD RMR alues we e also calcula ed, disca ding ei he (i) hose alues <85% o >115% o he mean 25 min RMR (low il e ), (ii) <90% o >110% o he mean 25 min RMR (medium il e ), Nu ien s 2020,12, 487 4 o 14 o (iii) <95% o >105% o he mean 25 min RMR (s ong il e ). Fo he minu es ha passed hese il e s, he means we e calcula ed o all pMVD alues a ailable. 2.5. Calcula ing he Res ing Me abolic and Nu ien Oxida ion Ra es Wei ’s equa ion (assuming ze o u ina y ni ogen exc e ion) [ 17 ] was used o calcula e RMR alues om he mean pMVD o VO 2 and VCO 2 ob ained wi h each gas exchange da a selec ion me hod. The FATOx and CHOOx a es we e calcula ed using F ayn’s s oichiome ic equa ions [ 18 ], also assuming ze o u ina y ni ogen exc e ion. Finally, he mean RMR, RQ, VO 2 , VCO 2 , FATOx, and CHOOx we e calcula ed o all gas exchange da a selec ion me hods unde each di e en condi ion. 2.6. Body Composi ion Body composi ion was de e mined by dual ene gy X- ay abso p iome y using a Disco e y Wi de ice (Hologic, Inc., Bed o d, MA, USA). Quali y con ols, he posi ioning o pa icipan s and analysis o he esul s we e pe o med acco ding o he manu ac u e ’s ecommenda ions. 2.7. S a is ical analysis Resul s a e p esen ed as mean ± SD unless o he wise s a ed. All analyses we e conduc ed using he S a is ical Package o he Social Sciences .22.0 (IBM SPSS S a is ics, IBM Co po a ion, Chicago, IL, USA). Signi icance was se a p<0.05. Repea ed-measu es analysis o a iance (ANOVA) wi h a pos -hoc Bon e oni es was used o de ec di e ences in RMR, RQ, VO 2 , VCO 2 , FATOx, and CHOOx es ima es ac oss he me hods o gas exchange da a selec ion. The CVs o VO 2 , VCO 2 , RQ, and VE ob ained ia he di e en me hods we e also compa ed. Two di e en ANOVA models we e used: one wi h ou le els o ixed ac o s (i.e., sho TI, long TI, SS , and il e ing), and one wi h 14 le els (all me hods and hei di e en condi ions). The di e ences be ween he me hods in e ms o he a iance in RMR explained by i s classical de e minan s (i.e., body weigh , body composi ion [lean and a masses], and sex) [ 13 ] we e examined by ei he simple linea eg ession (associa ions be ween RMR and body weigh ) o mul iple linea eg ession (associa ions be ween RMR and sex and body weigh ; RMR and sex and lean and a masses). 3. Resul s Table 1p o ides desc ip i e da a o he subjec s in bo h coho s. Table 1. Subjec desc ip i e cha ac e is ics. Young Adul s (n=107) Middle-Aged Adul s (n=74) Min Max Pe cen ile 10–90 Min Max Pe cen ile 10–90 Age (yea s) 22.2 ±2.2 18.2 26.6 19.1–25.2 53.5 ±5.3 45.0 66.0 47.0–61.7 Sex (n%) Women 72, 67 39, 53 35, 47 Men 35, 33 Me abolic ca used (n%) CCM Exp ess 46, 43 61, 57 69.3 ±15.9 167.8 ±8.7 24.5 ±4.4 79.8 ±13.2 41.4 ±9.5 24.0 ±8.9 35.0 ±7.8 0, 0 CPX Ul ima Ca diO2 74, 100 Body weigh (kg) 45.0 118.5 52.2–90.6 75.7 ±15.0 167.8 ±9.8 26.7 ±3.8 95.1 ±11.7 43.5 ±11.7 30.0 ±8.4 39.9 ±9.1 50.6 110.7 57.8–94.6 Heigh (cm) 148.5 195.1 157.2–180 148.3 189.8 155.8–181.6 BMI (kg/m2)17.2 38.4 19.4–30.9 19.0 38.0 22.0–31.7 Wais ci cum e ence (cm) 58.0 125.6 65.0–97.8 68.6 118.7 79.2–107.8 Lean mass (kg) 28.1 66.8 31.0–55.2 22.7 63.6 30.5–59.6 Fa mass (kg) 9.9 51.7 14.6–36.4 14.5 55.8 20.6–40.7 Fa mass (%) 15.3 51.9 26.2–44.4 23.0 59.4 26.7–51.1 Da a a e p esen ed as mean ±SD unless o he wise s a ed. Nu ien s 2020,12, 487 5 o 14 3.1. In luence o he Gas Exchange Da a Selec ion Me hod on Es ima es o RMR, RQ and Nu ien Oxida ion Figu e 1shows he RMR and RQ es ima es yielded by he gas exchange da a selec ion me hods o bo h he young and middle-aged adul s. In he young adul s, he sho and long TI me hods p o ided highe mean RMR es ima es han ei he he SS o il e ing me hods ( aking all condi ions oge he ; pos -hoc Bon e oni p<0.001; Figu e 1A). In he middle-aged adul s hey also p o ided highe mean RMR es ima es han he il e ing me hod ( aking all condi ions oge he ; pos -hoc Bon e oni p<0.001; Figu e 1B). No di e ences we e seen be ween he RMR es ima es yielded by he sho and long TIs, no be ween he SS and he il e ing me hods ( aking all condi ions oge he ) in ei he he young o he middle-aged adul s (all pos -hoc Bon e oni p=1.000; Figu e 1A,B). Fo he young adul s, he lowes mean RMR alues we e ob ained wi h he SS 4 min me hod (1440 kcal/day; Figu e 1E); howe e , he SS 4 min me hod was only s a is ically di e en om he TI 6–10 min and he TI 11–15 min. In he middle-aged adul s he lowes mean RMR alues we e p o ided by he s ong- il e me hod (1493 kcal/day; Figu e 1F); he s ong- il e me hod was s a is ically di e en om all he di e en me hods, wi h he excep ion o he TI 11–15 min, and he SS 3, 4, and 5 min condi ions. Table S1 shows he compa isons (i.e., pos -hoc Bon e oni) be ween he di e en me hods. Simila pa e ns we e obse ed when analyzing he in luence o gas exchange da a selec ion me hod on VO 2 and VCO 2 es ima es (Figu e S1). Las ly, he pe iods in which he SS we e achie ed (wi h he di e en SS me hods applied) in he young and he middle-aged adul s a e p esen ed in Figu e S2. We obse ed ha ~50% o young and middle-aged adul s achie ed hei SS s (i.e., he one p esen ing lowe mean CV) du ing he i s hal o he 30 min indi ec calo ime y assessmen . On he o he hand, we ound di e ences be ween he i s s eady s a e achie ed (i.e., he i s pe iod in which he CVs o VO 2 <10, VCO 2 <10, VE <10, and RQ <5) and he “bes ” SS achie ed (i.e., he pe iod wi h he lowes mean CVs) in RMR es ima ion in young adul s (Figu e S3). The RQ es ima es yielded by he sho TI me hod we e signi ican ly highe han all o he s in he young adul coho (all pos -hoc Bon e oni p<0.002; Figu e 1C) and ha il e ing me hod ( aking all condi ions oge he ) in he middle-aged adul s coho (pos -hoc Bon e oni p=0.038; Figu e 1D). Mo eo e , he long TI me hod p o ided highe RQ es ima es han he SS and il e ing me hods ( aking all condi ions oge he ) in he young adul (bo h Bon e oni pos -hoc p<0.013; Figu e 1C). No di e ences in RQ es ima es we e seen when compa ing he SS and il e ing me hods ( aking all condi ions oge he ) in ei he he young o he middle-aged adul s (pos -hoc Bon e oni p=1.000; Figu e 1C,D). Fu he mo e, he long TI and he SS we e no signi ican ly di e en han ei he he sho TI me hod o he il e ing me hods ( aking all condi ions oge he ) in he middle-aged adul s (Figu e 1D). The lowes mean RQ alues we e ob ained when using he s ong- il e me hod (0.84) in he young adul s (Figu e 1G). Howe e , he s ong- il e me hod was only s a is ically di e en om he TI 26–30 min and he TI 6–30 min. The lowes mean RQ alues we e ob ained when using he SS 3 min me hod (0.80) in he middle-aged adul s (Figu e 1H). Howe e , no s a is ical di e ences we e obse ed. As expec ed, he da a selec ion me hods yielding highe RQ es ima es also p o ided highe CHOOx and lowe FATOx es ima es, and ice e sa (Figu e 2). Nu ien s 2020,12, 487 6 o 14 Nu ien s 2020, 12, 487 6 o 14 Figu e 1. Di e ences among gas exchange da a selec ion me hods wi h espec o es ing me abolic a e (RMR) and espi a o y quo ien (RQ) es ima es. Black columns ep esen sho ime in e al (TI) pe iods (i.e., he means o he pe minu e en ila ion da a (pMVD]) alues o all a iables a ailable o hese ime pe iods, panels A–D; he pMVD alues o each sho TI pe iod, panels E,F). Ligh g ey columns ep esen long TI pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese ime pe iods, panels A–D; he means o he pMVD alues o each long TI pe iod, panels E,F). Whi e columns ep esen s eady s a e (SS ) pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese SS pe iods, panels A–D; he means o he pMVD alues o each SS pe iod, panels E,F). Da k g ey columns ep esen il e ing me hods (i.e., he means o he pMVD alues o all a iables a ailable o hese il e ing pe iods, panels A–D; he means o he pMVD alues o each il e ing pe iod, panels E,F). p- alues come om epea ed-measu es analysis o a iance (ANOVA). Iden ical indica o y le e s highligh di e ences as de e mined by pos -hoc Bon e oni analysis. Da a a e p esen ed as mean and s anda d e o o he mean (SEM). Min: minu es; VCO2: p oduc ion o ca bon dioxide; VO2: consump ion o oxygen. Figu e 1. Di e ences among gas exchange da a selec ion me hods wi h espec o es ing me abolic a e (RMR) and espi a o y quo ien (RQ) es ima es. Black columns ep esen sho ime in e al (TI) pe iods (i.e., he means o he pe minu e en ila ion da a (pMVD]) alues o all a iables a ailable o hese ime pe iods, panels A – D ; he pMVD alues o each sho TI pe iod, panels E , F ). Ligh g ey columns ep esen long TI pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese ime pe iods, panels A – D ; he means o he pMVD alues o each long TI pe iod, panels E,F ). Whi e columns ep esen s eady s a e (SS ) pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese SS pe iods, panels A – D ; he means o he pMVD alues o each SS pe iod, panels E , F ). Da k g ey columns ep esen il e ing me hods (i.e., he means o he pMVD alues o all a iables a ailable o hese il e ing pe iods, panels A–D; he means o he pMVD alues o each il e ing pe iod, panels E , F ). p- alues come om epea ed-measu es analysis o a iance (ANOVA). Iden ical indica o y le e s highligh di e ences as de e mined by pos -hoc Bon e oni analysis. Da a a e p esen ed as mean and s anda d e o o he mean (SEM). Min: minu es; VCO 2 : p oduc ion o ca bon dioxide; VO2: consump ion o oxygen. Nu ien s 2020,12, 487 7 o 14 Nu ien s 2020, 12, 487 7 o 14 The RQ es ima es yielded by he sho TI me hod we e signi ican ly highe han all o he s in he young adul coho (all pos -hoc Bon e oni p < 0.002; Figu e 1C) and ha il e ing me hod ( aking all condi ions oge he ) in he middle-aged adul s coho (pos -hoc Bon e oni p = 0.038; Figu e 1D). Mo eo e , he long TI me hod p o ided highe RQ es ima es han he SS and il e ing me hods ( aking all condi ions oge he ) in he young adul (bo h Bon e oni pos -hoc p < 0.013; Figu e 1C). No di e ences in RQ es ima es we e seen when compa ing he SS and il e ing me hods ( aking all condi ions oge he ) in ei he he young o he middle-aged adul s (pos -hoc Bon e oni p = 1.000; Figu e 1C,D). Fu he mo e, he long TI and he SS we e no signi ican ly di e en han ei he he sho TI me hod o he il e ing me hods ( aking all condi ions oge he ) in he middle-aged adul s (Figu e 1D). The lowes mean RQ alues we e ob ained when using he s ong- il e me hod (0.84) in he young adul s (Figu e 1G). Howe e , he s ong- il e me hod was only s a is ically di e en om he TI 26–30 min and he TI 6–30 min. The lowes mean RQ alues we e ob ained when using he SS 3 min me hod (0.80) in he middle-aged adul s (Figu e 1H). Howe e , no s a is ical di e ences we e obse ed. As expec ed, he da a selec ion me hods yielding highe RQ es ima es also p o ided highe CHOOx and lowe FATOx es ima es, and ice e sa (Figu e 2). Figu e 2. Di e ences among gas exchange da a selec ion me hods wi h espec o a oxida ion (FATOx) and ca bohyd a e oxida ion (CHOOx) a es. Black columns ep esen sho ime in e al (TI) pe iods (i.e., he means o he pe minu e en ila ion da a [pMVD] alues o all a iables a ailable o hese ime pe iods, panels A – D ; he pMVD alues o each sho TI pe iod, panels E , F ). Ligh g ey columns ep esen long TI pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese ime pe iods, panels A – D ; he means o he pMVD alues o each long TI pe iod, panels E , F ). Whi e columns ep esen s eady s a e (SS ) pe iods (i.e., he means o he pMVD alues o all a iables a ailable o hese SS pe iods, panels A – D ; he means o he pMVD alues o each SS pe iod, panels E , F ). Da k g ey columns ep esen il e ing me hods (i.e., he means o he pMVD alues o all a iables a ailable o hese il e ing pe iods, panels A – D ; he means o he pMVD alues o each il e ing pe iod, panels E , F ). p- alues come om epea ed-measu es analysis o a iance (ANOVA). Iden ical indica o y le e s highligh di e ences as de e mined by pos -hoc Bon e oni analysis. Da a a e p esen ed as mean and s anda d e o o he mean (SEM). Min: minu es. Nu ien s 2020,12, 487 8 o 14 3.2. Di e ences be ween he Me hods in Te ms o he Va iance in RMR Explained by I s Classical De e minan s The a iance in RMR explained by body weigh ( aking all condi ions oge he ) was 36%, 36%, 34% and 38% o he sho TI, long TI, SS , and il e ing me hods espec i ely in young adul s, and 50%, 51%, 52% and 51% espec i ely in he middle-aged adul s. The mos explained a iance was ob ained using he low- il e me hod (40%) in young adul s and he TI 6–10 min me hod (54%) in he middle-aged adul s. The a iance explained inc eased o 34–45% and 54–68% in he young and middle-aged adul s espec i ely a e including subjec sex in he model (Table 2). The mos explained a iance was ob ained wi h he low- il e me hod in young adul s, and bo h he TI 21–25 min and he medium- il e me hods in he middle-aged adul s. Howe e , li le di e ence was seen among he me hods in e ms o he a iance explained by he classical de e minan s o RMR (Table 2). A u he model including subjec sex, lean, and a masses inc eased he a iance in RMR explained by ~5% in he young adul s, bu no in he middle-aged adul s (Table 3). The low- il e me hod explained he g ea es a iance in RMR in he young adul s, and he TI 21–25 min me hod did so in he middle-aged adul s. Howe e , once again, li le di e ence was seen among he me hods in e ms o he a iance explained by he classical de e minan s o RMR (Table 3). Nu ien s 2020,12, 487 9 o 14 Table 2. Va iance in es ing me abolic a e (RMR) explained by sex and body weigh in each o he gas exchange da a selec ion me hods. Me hod o Da a Selec ion Young Adul s (n=107) Middle-Aged Adul s (n=74) Sex Weigh (kg) Sex Weigh (kg) Model R2Cons an βpβpModel R2Cons an βpβp TI 6–10 min 0.44 1232 −171.6 0.001 7.8 <0.001 0.67 1503 −424.0 0.001 8.9 0.001 TI 11–15 min 0.40 1340 −214.4 <0.001 7.3 <0.001 0.67 1616 −418.5 0.001 7.1 0.004 TI 16–20 min 0.40 1300 −214.5 <0.001 7.6 <0.001 0.60 1758 −445.2 0.001 6.2 0.031 TI 21–25 min 0.36 1342 −199.5 0.001 6.7 <0.001 0.68 1935 −507.5 0.001 4.9 0.051 TI 26–30 min 0.34 1208 −155.7 0.009 7.5 <0.001 0.54 1754 −409.8 0.001 5.5 0.058 TI 6–30 min 0.42 1284 −191.2 0.001 7.4 <0.001 0.66 1713 −441.0 0.001 6.5 0.010 TI 6–25 min 0.43 1303 −200.0 <0.001 7.3 <0.001 0.67 1703 −448.8 0.001 6.8 0.008 SS 3 min 0.39 1245 −194.9 0.001 7.8 <0.001 0.66 1669 −456.7 0.001 7.1 0.008 SS 4 min 0.39 1169 −167.5 0.004 7.9 <0.001 0.67 1786 −481.1 0.001 6.4 0.016 SS 5 min 0.36 1301 −207.0 0.001 7.1 <0.001 0.66 1763 −464.1 0.001 6.2 0.016 SS 10 min 0.41 1232 −182.8 0.001 7.7 <0.001 0.66 1681 −443.8 0.001 7.0 0.008 Low- il e 0.45 1188 −170.6 0.001 8.1 <0.001 0.67 1701 −442.9 0.001 6.6 0.009 Medium- il e 0.43 1209 −170.8 0.001 7.8 <0.001 0.68 1725 −447.5 0.001 6.4 0.010 S ong- il e 0.44 1218 −182.0 0.001 7.7 <0.001 0.67 1684 −431.7 0.001 6.2 0.012 Uns anda dized be a and p- alues (signi ican alues in bold) om mul iple eg ession analyses, in which sex and body weigh we e included as independen a iables, and he RMR es ima es yielded by he di e en me hods o gas exchange da a selec ion we e included as dependen a iables. Sex: 1 =men; 2 =women.