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Draft 1 Resting metabolic rate in muscular physique athletes: validity of existing methods and development of new prediction equations Grant M. Tinsley*, Austin J. Graybeal, M. Lane Moore Affiliation for all authors: Department of Kinesiology & Sport Management, Texas Tech University, Lubbock, TX, USA *Corresponding author: Grant M. Tinsley, Department of Kinesiology & Sport Management, Texas Tech University, Lubbock, TX, 79424, USA. grant.tinsley@ttu.edu (806) 834-5895. https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. mailto:grant.tinsley@ttu.edu Draft 2 Abstract Estimation of resting metabolic rate (RMR) is an important step for prescribing an individual’s energy intake. The purpose of this study was to evaluate the validity of portable indirect calorimeters and RMR prediction equations in muscular physique athletes. Twenty-seven males (n=17; BMI: 28.8±2.0 kg/m2; body fat: 12.5±2.7%) and females (n=10; BMI: 22.8±1.6 kg/m2; body fat: 19.2±3.4%) were evaluated. The reference RMR value was obtained from the ParvoMedics TrueOne® 2400 indirect calorimeter, and the Cosmed FitmateTM and Breezing® Metabolism Tracker provided additional RMR estimates. Existing RMR prediction equations based on body weight (BW) or dual-energy x-ray absorptiometry (DXA) fat-free mass (FFM) were also evaluated. Errors in RMR estimates were assessed using validity statistics, including t- tests with Bonferroni correction, linear regression, and calculation of the SEE, total error, and 95% limits of agreement. Additionally, new prediction equations based on BW (RMR [kcal/d] = 24.8*BW [kg] + 10) and FFM (RMR [kcal/d] = 25.9*FFM [kg] + 284) were developed using stepwise linear regression and evaluated using leave-one-out cross-validation. Nearly all existing BW- and FFM-based prediction equations, as well as the Breezing® Tracker, did not exhibit acceptable validity and typically underestimated RMR. The ten Haaf (2014) and Cunningham (1980) FFM-based equations may produce acceptable RMR estimates, although the Cosmed FitmateTM and newly developed BW- and FFM-based equations may be most suitable for RMR estimation in male and female physique athletes. Future research should provide additional external cross-validation of the newly-developed equations in order to refine the ability to predict RMR in physique athletes. Keywords: resting energy expenditure, metabolism, bodybuilders, Harris-Benedict, Mifflin, body composition, Cunningham, ten Haaf, indirect calorimetry Page 1 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 3 Introduction Measurement or estimation of resting metabolic rate (RMR) is frequently the first step in prescribing energy intake, both in the general population and athletes (Thomas et al. 2016). Even in highly active individuals, RMR represents a substantial contribution to total daily energy expenditure (TDEE). While laboratory methods, namely indirect calorimetry, are commonly utilized for RMR measurement, most individuals rely on prediction equations to estimate RMR. Prediction equations based on body weight (BW) have been utilized for over 100 years (Harris and Benedict 1918), and numerous distinct equations are presently employed (Flack et al. 2016). However, due to the known differences in the metabolic activity of fat mass and fat-free mass (FFM), several other equations predict RMR based on FFM rather than BW (Hayes et al. 2002). A detailed analysis of energy expenditure at the organ/tissue level of the body demonstrated a linear relationship between FFM and RMR within the range of FFM typically observed in humans, and equations typically possess a slope that ranges from 19.7 to 24.5 and a positive intercept of approximately 200 to 700 kcal/day (Wang et al. 2000). In athletes, some advocate the use of FFM-based prediction equations due to the relatively greater proportion of FFM in these individuals (ten Haaf and Weijs 2014). Several investigations have examined the validity of BW- or FFM-based equations in athletic populations, with some leading to the development of new athlete-specific equations. These investigations have examined a variety of athletic groups, including endurance athletes (Thompson and Manore 1996), a mixed group of athletes including waterpolo, judo and karate (De Lorenzo et al. 1999), rowers and canoeists (Carlsohn et al. 2011), a variety of team sport athletes (i.e. football, track and field, baseball, swimming and soccer) (Jagim et al. 2017) and a mixed group of individual and team sport athletes (ten Haaf and Weijs 2014). While some of the aforementioned prediction Page 2 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 4 equations were developed in narrowly defined groups of athletes, others included substantial heterogeneity in an attempt to produce a generalizable equation. It is recognized that the accuracy of RMR prediction equations may be population-specific, indicating that these equations cannot be indiscriminately applied groups that are dissimilar to those in which they were developed (da Rocha et al. 2005). Additionally, although generalizable equations are convenient, they may mask actual differences between specific sub-populations of athletes, who may vary in body composition and training practices that could impact RMR. For example, the groups of athletes included in the aforementioned equations do not typically exhibit the degree of muscularity observed in competitive physique athletes, whereas physique athletes may have lower energy intake and TDEE than athletes in some traditional sports (Slater and Phillips 2011). Proper prescription of energy intake to facilitate fat loss, while promoting the retention of FFM and physical performance, is a major goal of physique athletes preparing for competition (Helms et al. 2014). However, most of these athletes do not have access to traditional indirect calorimeters for measurement of RMR. Currently, there are several portable indirect calorimeters available, which may be a more accessible option to this athletic population. However, limited information is available concerning the validity of these devices for RMR estimation in athletes, and the price and complexity of the devices varies widely. Despite the existence of these portable devices, the most common method of RMR estimation remains the use of prediction equations. It is unclear if FFM-based equations, which necessitate the estimation of body composition, are superior to BW-based equations in this population. Theoretically, FFM-based equations could be advantageous, although this has not previously been examined. To our knowledge, no previous investigations have examined the validity of portable indirect calorimetry and BW- or FFM-based RMR prediction equations in physique athletes. Therefore, Page 3 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l use o nl y. Draft 5 the purpose of this study was to evaluate the utility of several practical methods of RMR estimation in male and female physique athletes and to produce preliminary RMR prediction equations for this population. Materials and Methods At a single research visit, the RMR of physique athletes was measured via three indirect calorimeters. Body weight and composition were also assessed to allow for estimation of RMR using prediction equations. Twenty-seven physique athletes volunteered to participate in this study as previously described (Graybeal et al. 2018). To be eligible for inclusion in this analysis, participants were required to meet at least one of the following criteria: 1) have competed in a bodybuilding or physique competition within the last year; 2) have plans to compete within the next year; or 3) self-identify as a bodybuilder and exhibit a physique commensurate with competitive physique athletes, as evaluated by study investigators. Additionally, prospective participants were required to be between the ages of 18 and 50, generally healthy, and report the completion of ≥ 3 sessions per week of resistance training, continuously for ≥ 3 years, prior to screening. This study was approved by the Texas Tech University institutional review board, and all participants signed the informed consent document prior to participation. Participants reported to the laboratory in the morning after an overnight (≥ 8 hours) abstention from food, fluid, supplement or medication ingestion, and exercise. Body weight and height were assessed using a digital scale and stadiometer (Seca 769, Hamburg, Germany). FFM was estimated via dual-energy x-ray absorptiometry (DXA). DXA scans were performed on a calibrated GE Lunar Prodigy scanner with enCORE software (v. 16.2), and participant positioning was conducted according to manufacturer recommendations. Due to the large body Page 4 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 6 size of many participants, it was necessary to perform the reflection scanning technique in which the unobserved portion of the body (i.e. the left arm) is estimated from the observed portion of the body. This technique was performed in accordance with manufacturer recommendations and has been reported to induce minimal error (Moco et al. 2018; Tinsley et al. 2018b). In order to further reduce potential errors caused by this procedure, scans were conducted in duplicate and averaged for analysis. DXA lean soft tissue and bone mineral content were summed to provide an estimate of FFM. Following body composition assessment, RMR was assessed by three indirect calorimetry devices. The TrueOne® 2400 (ParvoMedics, Sandy, UT, USA) was designated as the reference method, and additional methods were a portable research-grade device (FitmateTM, Cosmed, Rome, Italy) and a portable consumer-grade device (Breezing® Metabolism Tracker, Breezing, Tempe, AZ, USA). The TrueOne® 2400 was selected as the reference method due to its demonstrated accuracy (Cooper et al. 2009; Kaviani et al. 2018). A recent study evaluated the accuracy and reliability of 12 indirect calorimeters using methanol combustion (Kaviani et al. 2018). Of the 12 devices, two separate TrueOne® 2400 systems were ranked 1st and 2nd for CO2 recovery, 2nd and 5th for O2 recovery and 2nd and 4th for RER accuracy. Furthermore, both of the evaluated TrueOne® 2400 units measured CO2 recovery, O2 recovery and RER within 2% of theoretical values, unlike most other devices. The TrueOne® 2400 unit used in this study was less than 2 years old at study commencement, and regular maintenance and calibrations were performed according to manufacturer instructions throughout this time period. A new Cosmed FitmateTM device was purchased for this study, and the oxygen sensor remained in the “optimal” state throughout data collection. A new Breezing® Tracker device was purchased from the manufacturer approximately 3 months prior to study commencement, and all testing was Page 5 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 7 completed in less than 7 months after receipt of the device and its associated supplies (e.g. sensor cartridges). Pre-assessment standardization and testing were conducted according recommended procedures (Compher et al. 2006). Briefly, the participant was rested and fasted overnight prior to each assessment and was instructed to remain motionless, but awake, throughout testing. Each participant was offered a blanket at each assessment in order to promote a comfortable body temperature, and all testing took place in the same climate-controlled room with the lights dimmed. Due to previous laboratory assessments conducted at the research visit, each participant rested in the supine position for approximately 30 minutes prior to the commencement of the first RMR assessment. The order of RMR assessments was randomly determined using the random integer set generator available at random.org. For each device, manufacturer procedures were followed. RMR via TrueOne® 2400 (RMRPARVO) and FitmateTM (RMRCOSMED) was assessed in the supine position, while RMR via Breezing® (RMRBREEZING) was assessed in the seated position per manufacturer instructions. Regardless of assessment order, each participant moved from the supine to seated position for a period of approximately two minutes between RMR assessments. Prior to TrueOne® 2400 assessments, daily gas and flow calibrations were performed. Prior to FitmateTM assessments, daily flow and oxygen sensor calibrations were performed. For both the TrueOne® 2400 and FitmateTM assessments, the first five minutes of each test were discarded, and the assessment continued until there was a period of 5 consecutive minutes with a coefficient of variation (CV) for RMR of ≤ 10%. Using 1-minute averaging, the average CVs in this study were 4.2 ± 1.5% and 4.6 ± 1.9% for RMRPARVO and RMRCOSMED, respectively. The Breezing® device utilizes a sensor cartridge and flow meter to evaluate expired air as previously Page 6 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 8 described (Xian et al. 2015). The device is synced with a phone or tablet, and each single-use sensor has a QR code that is scanned by the associated phone or tablet to provide calibration information for the sensor. During each assessment, the participant breathes through a disposable mouthpiece for 1 to 2 minutes, until 6 L of air has been expired during the assessment. RMR is then estimated from VO2 and VCO2 using the Weir equation (Xian et al. 2015) and reported in kcal/d. In addition to the indirect calorimetry assessments, RMR was predicted via five BW- based equations, five FFM-based equations and one organ/tissue-based equation (Table 1). BW- based equations (Harris and Benedict 1918; FAO 1985; Mifflin et al. 1990; De Lorenzo et al. 1999; ten Haaf and Weijs 2014) utilized BW obtained on a digital scale (Seca 769, Hamburg, Germany), while FFM-based equations (Cunningham 1980; Owen et al. 1987; Mifflin et al. 1990; Cunningham 1991; ten Haaf andWeijs 2014) utilized DXA FFM, and the organ/tissue model used various components of DXA output as previously described (Hayes et al. 2002). Statistical Analysis Potential differences in RMR between the reference method and alternative methods were analyzed using dependent t-tests with a Bonferroni-adjusted alpha level due to multiple comparisons (p ≤ 0.0033). The constant error (CE) was determined as the mean difference between an alternate RMR assessment and the reference method (e.g., RMRALTERNATE – RMRPARVO). Additionally, the Pearson product moment correlation coefficient (r), coefficient of determination (R2), standard error of the estimate (SEE), and total error (TE) were calculated. The TE, also known as the root mean square error (RMSE), was calculated as: 𝑇𝐸 = Σ(𝑅𝑀𝑅𝑃𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 ― 𝑅𝑀𝑅𝑃𝐴𝑅𝑉𝑂)2/𝑛 Page 7 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 9 The TE represents the average deviation of individual scores from the line of identity between the reference method and each alternative method, whereas the SEE indicates the deviation of individual data points around the line of best fit for the reference method and each alternative method (Heyward and Wagner 2004). The following thresholds were used to describe the r values: trivial (<0.1), small (0.1 to 0.29), moderate (0.30 to 0.49), large (0.50 to 0.69), very large (0.70 to 0.89), and extremely large or “near perfect” (0.90 to 1.00) (Hopkins et al. 2009). The effect size (ES) of the differences between methods was determined using Cohen’s d. The magnitude of the ES was interpreted as: very small (<0.2), small (0.2 – 0.59), moderate (0.6 – 1.19), large (1.2 – 2.0), and very large (>2.0) (Hopkins, Marshall et al. 2009). The Bland-Altman method (Bland and Altman 1986) was used to identify the 95% limits of agreement (LOA) between the reference and alternative methods. The 95% LOA indicate the individual predictive accuracy of a method based on a 95% confidence interval. Linear regression was utilized to evaluate proportional bias between the reference method and alternative methods (i.e. varying discrepancies between reference and alternative methods based on RMR values) as previously described (Tinsley 2017). Additionally, stepwise linear regression was utilized to develop RMR prediction equations from relevant variables (i.e. BW, FFM, age, sex and height). Due to the relatively small sample size, leave-one-out cross-validation was utilized to evaluate the newly developed equations (Ivanescu et al. 2016). This procedure involves sequentially removing each participant’s data, developing linear regression equations using the remaining data, and calculating the error produced when the regression equations are applied to the excluded data. The TE (i.e. RMSE) of the leave-one-out analysis was calculated using the prediction errors (i.e. CE) observed when regression equations were applied to excluded data. These leave-one-out TE values were compared to the TE values of the regression equations developed in the entire Page 8 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 10 sample (Lohman et al. 2000). Data were analyzed using IBM SPSS (v. 25) and Microsoft Excel (v. 16.11). Results All participants self-identified as bodybuilders, with 48% reporting participation in a physique contest in the past year. All participants reported practicing high-volume resistance training for ≥ 3 years, with current training of 5.7 ± 0.9 days per week. The rates of self-reported anabolic androgenic steroid (AAS) use were 26% (M: 35%, F: 10%) for current use and 41% (M: 59%, F: 10%) for use in the previous 3 years. However, it is believed that under-reporting of AAS usage may have occurred. The DXA fat-free mass index (FFMI) of male participants (24.2 ± 1.3 kg/m2) was approximately 2 SD greater than reference values from the National Health and Nutrition Examination Survey (NHANES), and the DXA FFMI of female participants (17.7 ± 0.9 kg/m2) was approximately 1 SD greater than NHANES reference values (Kelly et al. 2009). Conversely, the DXA fat-mass index (FMI) of male and female participants (M: 3.6 ± 0.9 kg/m2; F: 4.4 ± 1.0 kg/m2) was approximately 1 SD below NHANES reference values (Kelly et al. 2009). Participant characteristics are displayed in Table 2. Validity of the evaluated RMR methods for males and females combined are presented in Table 3, while individual results for males and females are presented in Tables 4 and 5, respectively. RMRCOSMED was not significantly different from RMRPARVO in males, females or males and females combined (+3.0 to 4.3%; trivial to small ES). Additionally, proportional bias was not present, and the LOAs were narrow relative to other methods (Figure 1A). In contrast, RMRBREEZING was 14.5% lower (moderate ES) in males and females combined when compared to RMRPARVO. The level of disagreement was much larger in males (-22.0%; large ES) than in Page 9 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 11 females (+4.3%; small ES). However, in both males and females, LOAs for RMRBREEZING were wide relative to other methods (Figure 1B). In general, BW-based equations underestimated RMR in males and females combined by 4.5 to 15.1% (small to moderate ES) (Figure 2; Table 3). In males, all BW-based equations underestimated RMR by ≥ 10.1% (medium to large ES), with the exception of the ten Haaf equation, which did not differ significantly from the reference method (-6.2%; small ES). However, all BW-based equations demonstrated statistically significant negative proportional bias with regression coefficients of ≥ -0.65. In females, RMR estimates from BW equations were not statistically different from the reference method. However, three BW-based equations underestimated RMR by ≥ 7.1% (moderate to large ES), while one overestimated RMR by 7.1% (DeLorenzo; moderate ES) and one (ten Haaf) displayed no CE (trivial ES). Although not statistically significant, BW-based equations demonstrated possible negative proportional bias, with regression coefficients varying from -0.28 to -1.0. In males and females combined, three FFM-based equations underestimated RMR by 10.2 to 17.4% (small to moderate ES), while two equations (Cunningham [1980] and ten Haaf) did not differ significantly from RMRPARVO (-0.8 to -2.6%; trivial ES) and exhibited relatively low TE (Figure 3). In males, three FFM-based equations underestimated RMR by 10.9 to 15.5% (moderate ES), while two other equations (ten Haaf, Cunningham [1980]) exhibited underestimations of RMR relative to RMRPARVO (-2.0 to 3.9%; trivial to small ES) that were not statistically significant. Statistically significant negative proportional bias was seen for all FFM- based equations in males (Table 4). In females, two FFM-based equations significantly underestimated RMR by 11.7 to 16.8% (moderate to very large ES), while three equations exhibited deviations (-8.6 to +2.2%; trivial to moderate ES) that were not statistically significant. Page 10 of 37 https://mc06.manuscriptcentral.com/apnm-pubs AppliedPhysiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 12 Although not statistically significant, all FFM-based equations demonstrated possible negative proportional bias, with regression coefficients ≥ -0.24 (Table 5). For males, females, and the entire sample, RMR estimated by the organ/tissue equation (RMRHAYES; Figure 1C) did not outperform several predictive equations based solely on FFM or BW (Tables 3 – 5). New BW- and FFM-based RMR prediction equations were developed within the entire sample (Figure 4). In the BW-based model (BWTINSLEY), BW was the only statistically significant predictor of RMR (B: 24.78; p < 0.0001). Excluded variables included sex (B: 0.094; p = 0.57), age (B: -0.035; p = 0.66) and height (B: -0.070; p = 0.68). Overall, BWTINSLEY had an r of 0.921 and R2 of 0.849. The final BWTINSLEY equation is: , with RMR 𝑅𝑀𝑅 = 24.8 ∗ 𝐵𝑊 + 10 calculated in kcal/d and BW in kg. In the FFM-based model (FFMTINSLEY), DXA FFM was the only statistically significant predictor of RMR (B: 25.94; p < 0.0001). Excluded variables included sex (B: -0.066; p = 0.73), age (B: 0.005; p = 0.95), height (B: -0.115; p = 0.52) and weight (B: 0.417; p = 0.37). Overall, the FFM-based model had an r of 0.923 and R2 of 0.851. The final FFMTINSLEY equation is: , with RMR calculated in kcal/d 𝑅𝑀𝑅 = 25.9 ∗ 𝐹𝐹𝑀 + 284 and FFM in kg. In males, females and the entire sample, the average leave-one-out TE for both the BW- and FFM-based equations were ≤ 15 kcal/d higher than the TEs when linear regression was performed using the entire dataset (Table 6). The newly developed BWTINSLEY equation generally minimized CE, ES, TE, and LOA relative to other BW-based equations. Additionally, it was the only model without statistically significant proportional bias (Figure 2F). The newly developed FFMTINSLEY equation also generally minimized CE, ES, TE, and LOA relative to other FFM-based equations and exhibited less proportional bias (Figure 3F). Additional sub-analysis with the newly developed equations was performed on participants reporting current AAS usage (6 M, 1 F) versus those reporting no current usage of Page 11 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 13 AAS. Although these results should be interpreted with caution due to the self-reported nature of AAS usage and the substantial differences in sample size between those reporting current AAS usage (n=7) and those reporting no current AAS usage (n=20), the newly developed equations produced CE and r values of similar magnitudes in users and non-users when compared to the reference method. When using the BWTINSLEY equation, the CE was 29 kcal/d (r = 0.93) in non- users vs. – 32 kcal/d (r = 0.88) in users. Additionally, there was no difference in CE when users and non-users were compared via independent samples t-test (p = 0.44). When using the FFMTINSLEY equation, the CE was 9 kcal/d (r = 0.92) in non-users vs. -36 kcal/d (r = 0.91) in users. Additionally, there was no difference in CE when users and non-users were compared via independent samples t-test (p = 0.57). Discussion The purpose of this study was to evaluate the utility of several practical methods of RMR estimation in male and female physique athletes, including two portable indirect calorimeters and several commonly used BW- and FFM-based prediction equations. Additionally, preliminary RMR prediction equations based on BW or FFM were developed for this population. The major finding of this study was that the Cosmed FitmateTM portable indirect calorimeter and the newly developed BW- and FFM-based prediction equations produced less error than all other methods for RMR estimation in male and female physique athletes. No other BW-based RMR prediction equations performed acceptably in males, although the ten Haaf (2014) BW equation produced relatively low errors in females. The existing Cunningham (1980) and ten Haaf (2014) FFM- based equations produced fairly accurate RMR estimates in males and females, although substantial proportional bias was present, particularly when examining males alone. All other Page 12 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 14 RMR prediction equations evaluated in this study are apparently unsuitable for use in muscular physique athletes. For this investigation, the ParvoMedics TrueOne® 2400 indirect calorimeter was designated as the reference method of assessment due to its documented accuracy and reliability (Cooper et al. 2009; Kaviani et al. 2018). Of the methods evaluated in this study, the Cosmed FitmateTM portable indirect calorimeter produced RMR estimates with the narrowest 95% LOA and highest correlation when compared to the reference RMR. The Cosmed FitmateTM and the newly developed BW- and FFM-based equations exhibited relatively low TE and generally exhibited smaller regression coefficients than most existing prediction equations, indicative of less proportional bias. The CE for the FitmateTM was similar in males in females (~69 kcal/d; trivial to small ES), and RMR estimates were not significantly different than the reference method. Previous research also supports the validity and reliability of the FitmateTM system (Nieman et al. 2006; Vandarakis et al. 2013). The other portable indirect calorimetry device evaluated in this study (Breezing® Tracker) utilizes a portable metabolic analyzer that was previously deemed to produce valid RMR estimates relative to the Douglas bag method (CE: 59 ± 31 kcal/d; 95% LOA: ±215 kcal/d) in a sample of 17 healthy adults, ranging from underweight to obese (Zhao et al. 2014). Another report derived from the same study found a CE of 6 kcal/d when >300 tests were performed in 12 adults across a range of energy expenditures from 1,000 to 4,000 kcal/d (Xian, Quach et al. 2015). However, despite these results, the Breezing® device performed very poorly in the present investigation. The device produced alarming underestimations of RMR in males (CE: -513 kcal/d; large ES), a trivial correlation with the reference method (r: 0.02), and wholly unacceptable LOA (±919 kcal/d). In females, Breezing® produced the same CE as Cosmed (67 kcal/d) but only a moderate correlation (r: 0.38) with the Page 13 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 15 reference method. Additionally, it produced a large regression coefficient (B: 0.569), indicative of proportional bias, and the widest LOA of any method in females. Overall, this device performed worse than all other methods in males, including prediction equations, as well as performing poorly in females. Based on these results, the use of this device for estimating RMR in this population is strongly discouraged. In males, most existing BW- and FFM-based equations substantially underestimated RMR by up to 393 kcal/d, while also exhibiting unacceptable levels of negative proportional bias(i.e. greater underestimation of RMR in individuals with higher RMR). Based on the results of this investigation, none of the existing BW-based equations can be deemed acceptable for use in male physique athletes. However, when applied to males, the newly developed BW-based equation demonstrated low error, a high correlation with the reference method, and no statistically significant proportional bias. Overall, the performance of the new BW-based equation was superior to other BW-based equations and similar to the Cosmed FitmateTM in males. When considering FFM-based equations, the Cunningham (1980) and ten Haaf (2014) FFM-based equations produced less error than other equations in males, with the exception of the newly-developed FFM-based equation. While FFMTINSLEY demonstrated a smaller magnitude of proportional bias than the other methods, statistically significant bias was still observed. The frequent negative proportional bias observed in males may be partially attributed to one participant with a very high RMR assessed by the reference method (2,998 kcal/d) that was consistently underestimated by prediction equations. When a FFM-based equation is utilized for RMR prediction in male physique athletes, the newly developed equation may be suitable. However, appropriate caution should be employed when interpreting RMR estimates, particularly in very large individuals with high RMR. Page 14 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 16 In females, the ten Haaf (2014) equation was the only existing BW-based prediction equation deemed acceptable for use. Both the ten Haaf and newly-developed BW-based equation can potentially be used in female physique athletes similar to those examined in this investigation. As in males, the Cunningham (1980) and ten Haaf (2014) equations produced lower error than other FFM-based equations. Overall, these two equations, along with the newly- developed FFM-based equation, may be acceptable for use in female physique athletes similar to those evaluated in the present study. It is noteworthy that, although the Cunningham (1980) FFM-based equation produced relatively low error in males and females, individuals who were trained athletes were specifically excluded during the development of this equation, and the researchers’ goal was to develop an equation for a population of normal adults (Cunningham 1980). Additionally, since body composition was not available for the participants used in equation development, it was predicted from the participants’ body mass and age. Therefore, the accuracy of this equation is likely serendipitous rather than due to similarities between the populations. A more recent equation developed by Cunningham et al. (1991) produced relatively higher error than the older equation in the present investigation. In contrast, ten Haaf et al. (2014) developed their BW- and FFM- based equations in a group of male and female athletes from a variety of sports, including long distance running and cycling, gymnastics, sprinting, rowing, swimming, fitness, hockey, soccer, volleyball, dancing, martial arts, skating and tennis. Despite the diverse nature of the participants used in equation development, the ten Haaf FFM-based prediction equation generally performed well in the present sample of physique athletes. Additionally, the BW-based equation performed well in females. While these results are promising for the application of the ten Haaf equations in diverse athletic populations, the new BWTINSLEY and FFMTINSLEY equations were developed for a Page 15 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 17 specific target population of athletes (i.e. muscular physique competitors) rather than broad application to athletes in general. Nonetheless, cross validation of the newly developed equations in sports with the most muscular athletes (e.g. American football) may be warranted. Within the participants of this study, the new BW- and FFM-based equations performed very similarly when compared to the reference RMR value. Although this indicates that either could be used in individuals similar to the participants of the present study, an argument can be made that the BW-based equation could be preferable for several reasons. First, the new FFM- based equation was developed using DXA FFM, and DXA is often unavailable for body composition assessment. The utilization of FFM estimates from other devices could impact this equations accuracy due to discrepancies between DXA and other methods in athletic populations (Moon et al. 2009; Graybeal et al. 2018). We performed additional analysis in our sample indicating that the magnitude of difference in RMR with FFM estimates from alternative methods (i.e. multi-compartment models, multiple bioelectrical impedance analysis devices and bioimpedance spectroscopy) ranges from -30 to +74 kcal/d on average (data not shown). Additionally, much lower assessment error can be expected when evaluating BW within a single individual as compared to FFM. For both body composition assessment and RMR predictions based on body composition, caution should be employed when evaluating a single individual due to the distinct possibility of over- or under-estimation of body compartments (e.g. FFM) in any given individual. Lastly, performing a simple BW measurement is much more feasible than accurate body composition assessment in most settings. For these reasons, the most practical option may be to employ the newly developed BW-based equation, provided that the individual being evaluated exhibits similar characteristics to those used for equation development in this population (Table 2). Page 16 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 18 In the present investigation, all participants except one reported that they were in their offseason from competitions or not currently in a specific phase of their competitive cycle. This may be attributable to the fact that the data collection for this study was performed in the fall (i.e. late August to mid-December). As such, our results are generalizable to physique athletes who are not currently in the preparatory phase prior to a competition. It has been documented that decreases in RMR are observed in the competition preparation periods, but that RMR is recovered relatively quickly after competition as energy intake increases (Trexler et al. 2017; Tinsley et al. 2018a). The self-reported nature of AAS usage and limited number of individuals reporting current AAS usage did not allow for comprehensive evaluation of possible differences between AAS users and natural competitors. However, our preliminary evaluation did not reveal appreciable differences in the performance of the newly developed equations in AAS users versus non-users. Based on the available information, it is believed that the developed equations can be used in athletes similar to those in our sample regardless of current AAS usage. There are several other limitations to the present investigation. Our sample size is small for the development ofnew predictive equations, although relatively few prospective participants were available for evaluation due to the special population being assessed. However, we performed leave-one-out cross-validation in accordance with recommendations for small samples sizes. Nonetheless, we encourage additional external cross-validation of our newly developed equations in order to more fully determine their utility. Our analysis did not reveal improved utility of separate RMR prediction equations for male and female physique athletes (data not shown). However, future research should continue to investigate whether a sex term improves prediction of RMR in athletes. Although all participants reported to the laboratory after an Page 17 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 19 overnight period of fasting and resting, it is possible that the frequent training sessions of the participants could have altered RMR due to prior exercise. However, this limitation was deemed necessary due to the unwillingness of prospective participants to abstain from exercise for longer periods of time. In this respect, we believe our sample is representative of physique athletes in general. In conclusion, the Cosmed FitmateTM portable indirect calorimeter and the newly developed BW- and FFM-based prediction equations may be suitable for RMR estimation in male and female physique athletes similar to those in the present study. Additionally, the ten Haaf (2014) and Cunningham (1980) FFM-based equations may be acceptable for use in male and female physique athletes, with the ten Haaf BW-based equation being suitable in females only. All other BW- and FFM-based prediction equations that were evaluated, as well as the Breezing® Tracker, do not exhibit acceptable validity and frequently underestimate RMR in physique athletes. Future research should externally cross-validate the developed equations in order to refine the ability to practically produce valid estimates of RMR in this unique athletic population. Page 18 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 20 Acknowledgements The authors would like to thank Megan Cruz, Alfred Kankam, and Michael Villarreal for their assistance with data collection for this study. Conflict of Interest Statement The authors have no conflicts of interest to report. Page 19 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 21 References Bland, J.M. and Altman, D.G. 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet, 1: 307-10. Carlsohn, A., Scharhag-Rosenberger, F., Cassel, M., and Mayer, F. 2011. 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DOI: 10.1093/ajcn/54.6.963 da Rocha, E.E., Alves, V.G., Silva, M.H., Chiesa, C.A., and da Fonseca, R.B. 2005. Can measured resting energy expenditure be estimated by formulae in daily clinical nutrition practice? Curr. Opin. Clin. Nutr. Metab. Care. 8: 319-28. DOI: 10.1097/01.mco.0000165012.77567.1e Page 20 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 22 De Lorenzo, A., Bertini, I., Candeloro, N., Piccinelli, R., Innocente, I., and Brancati, A. 1999. A new predictive equation to calculate resting metabolic rate in athletes. J. Sports Med. Phys. Fitness, 39: 213-9. PMID: 10573663 FAO 1985. Energy and protein requirements: Report of a Joint FAO/WHO/UNU Expert Consultation. Flack, K.D., Siders, W.A., Johnson, L., and Roemmich, J.N. 2016. Cross-Validation of Resting Metabolic Rate Prediction Equations. Journal of the Academy of Nutrition and Dietetics, 116: 1413-1422. DOI: 10.1016/j.jand.2016.03.018 Graybeal, A.J., Moore, M.L., Cruz, M.R., and Tinsley, G.M. 2018. Body Composition Assessment in Male and Female Bodybuilders: A 4-Compartment Model Comparison of Dual- Energy X-Ray Absorptiometry and Impedance-Based Devices. Journal of Strength & Conditioning Research, Published Ahead of Print. DOI: 10.1519/jsc.0000000000002831 Harris, J.A. and Benedict, F.G. 1918. A Biometric Study of Human Basal Metabolism. Proc. Natl. Acad. Sci. USA, 4: 370-3. PMID: 16576330 Hayes, M., Chustek, M., Wang, Z., Gallagher, D., Heshka, S., Spungen, A., et al. 2002. DXA: potential for creating a metabolic map of organ-tissue resting energy expenditure components. Obes. Res. 10: 969-77. DOI: 10.1038/oby.2002.132 Helms, E., Aragon, A., and Fitschen, P. 2014. Evidence-based recommendations for natural bodybuilding contest preparation: nutrition and supplementation. J. Int. Soc. Sports Nutr. 11. DOI: 10.1186/1550-2783-11-20 Heyward, V.H. and Wagner, D. 2004. Applied body composition assessment: Champaign, IL : Human Kinetics. Page 21 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 23 Hopkins, W.G., Marshall, S.W., Batterham, A.M., and Hanin, J. 2009. Progressive statistics for studies in sports medicine and exercise science. Med. Sci. Sports Exerc. 41: 3-13. DOI: 10.1249/MSS.0b013e31818cb278 Ivanescu, A.E., Li, P., George, B., Brown, A.W., Keith, S.W., Raju, D., et al. 2016. The importance of prediction model validation and assessment in obesity and nutrition research. Int. J. Obes. (Lond). 40: 887-94. DOI: 10.1038/ijo.2015.214 Jagim, A.R., Camic, C.L., Kisiolek, J., Luedke, J., Erickson, J., Jones, M.T., et al. 2017. The accuracy of resting metabolic rate prediction equations in athletes. Journal of Strength & Conditioning Research, DOI: 10.1519/jsc.0000000000002111 Kaviani, S., Schoeller, D. A., Ravussin, E., Melanson Edward, L., Henes Sarah, T., Dugas Lara, R., et al. 2018. Determining the Accuracy and Reliability of Indirect Calorimeters Utilizing the Methanol Combustion Technique.Nutrition in Clinical Practice, 33: 206-216. DOI: 10.1002/ncp.10070 Kelly, T.L., Wilson, K.E., and Heymsfield, S.B. 2009. Dual Energy X-Ray Absorptiometry Body Composition Reference Values from NHANES. PLoS ONE, 4: e7038. DOI: 10.1371/journal.pone.0007038 Lohman, T.G., Caballero, B., Himes, J.H., Davis, C.E., Stewart, D., Houtkooper, L., et al. 2000. Estimation of body fat from anthropometry and bioelectrical impedance in Native American children. International Journal Of Obesity, 24: 982. DOI: 10.1038/sj.ijo.0801318 Mifflin, M.D., Jeor, S.T.S., Hill, L.A., Scott, B.J., Daugherty, S.A., and Koh, Y.O. 1990. A new predictive equation for resting energy expenditure in healthy individuals. The Am. J. Clin. Nutr. 51: 241-247. DOI: 10.1093/ajcn/51.2.241 Page 22 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 24 Moco, A.V., Matias, C.N., Santos, D.A., Rocha, P.M., Minderico, C.S., Cyrino, E.S., et al. 2018. Usefulness of Reflection Scanning in Determining Whole-Body Composition in Broadly Built Individuals Using Dual-Energy X-ray Absorptiometry. J. Clin. Densitom. In Press. DOI: 10.1016/j.jocd.2018.03.007 Moon, J.R., Eckerson, J.M., Tobkin, S.E., Smith, A.E., Lockwood, C.M., Walter, A.A., et al. 2009. Estimating body fat in NCAA Division I female athletes: a five-compartment model validation of laboratory methods. European Journal of Applied Physiology, 105: 119-130. DOI: 10.1007/s00421-008-0881-9 Nieman, D.C., Austin, M.D., Benezra, L., Pearce, S., McInnis, T., Unick, J., et al. 2006. Validation of Cosmed's FitMate in measuring oxygen consumption and estimating resting metabolic rate. Res. Sports Med. 14: 89-96. DOI: 10.1080/15438620600651512 Owen, O.E., Holup, J.L., D'Alessio, D.A., Craig, E.S., Polansky, M., Smalley, K.J., et al. 1987. A reappraisal of the caloric requirements of men. Am. J. Clin. Nutr. 46: 875-85. DOI: 10.1093/ajcn/46.6.875 Slater, G. and Phillips, S.M. 2011. Nutrition guidelines for strength sports: Sprinting, weightlifting, throwing events, and bodybuilding. Journal of Sports Sciences, 29: S67-S77. DOI: 10.1080/02640414.2011.574722 ten Haaf, T. and Weijs, P.J.M. 2014. Resting Energy Expenditure Prediction in Recreational Athletes of 18–35 Years: Confirmation of Cunningham Equation and an Improved Weight- Based Alternative. PLoS ONE, 9. DOI: 10.1371/journal.pone.0108460 Thomas, D.T., Erdman, K.A., and Burke, L.M. 2016. American College of Sports Medicine Joint Position Statement. Nutrition and Athletic Performance. Med. Sci. Sports Exerc. 48: 543-68. DOI: 10.1249/MSS.0000000000000852 Page 23 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 25 Thompson, J. and Manore, M.M. 1996. Predicted and measured resting metabolic rate of male and female endurance athletes. J. Am. Diet. Assoc. 96: 30-4. DOI: 10.1016/s0002- 8223(96)00010-7 Tinsley, G.M. 2017. Proportional bias between dual-energy x-ray absorptiometry and bioelectrical impedance analysis varies based on sex in active adults consuming high- and low- carbohydrate diets. Nutr. Res. 42: 85-100. DOI: 10.1016/j.nutres.2017.05.003 Tinsley, G.M., Trexler, E.T., Smith-Ryan, A.E., Paoli, A., Graybeal, A.J., Campbell, B.I., et al. 2018a. Changes in Body Composition and Neuromuscular Performance Through Preparation, Two Competitions, and a Recovery Period in an Experienced Female Physique Athlete. Journal of Strength & Conditioning Research, Ahead of Print. DOI: 10.1519/JSC.0000000000002758 Tinsley, G.M., Moore, M.L., and Graybeal, A.J. 2018b. Precision of Dual-Energy X-Ray Absorptiometry Reflection Scans in Muscular Athletes. Journal of Clinical Densitometry, Ahead of Print. DOI: 10.1016/j.jocd.2018.09.005 Trexler, E.T., Hirsch, K.R., Campbell, B.I., and Smith-Ryan, A.E. 2017. Physiological Changes Following Competition in Male and Female Physique Athletes: A Pilot Study. Int. J. Sport Nutr. Exerc. Metab. 1-25. DOI: 10.1123/ijsnem.2017-0038 Vandarakis, D., Salacinski, A.J., and Broeder, C.E. 2013. A comparison of COSMED metabolic systems for the determination of resting metabolic rate. Res. Sports. Med. 21: 187-94. DOI: 10.1080/15438627.2012.757226 Wang, Z., Heshka, S., Gallagher, D., Boozer, C.N., Kotler, D.P., and Heymsfield, S.B. 2000. Resting energy expenditure-fat-free mass relationship: new insights provided by body composition modeling. Am. J. Physiol. Endocrinol. Metab. 279: E539-45. DOI: 10.1152/ajpendo.2000.279.3.E539 Page 24 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 26 Xian, X., Quach, A., Bridgeman, D., Tsow, F., Forzani, E., and Tao, N. 2015. Personalized indirect calorimeter for energy expenditure (EE) measurement. Global Journal of Obesity, Diabetes and Metabolic Syndrome, 2: 004-008. DOI: 10.17352/2455-8583.000007 Zhao, D., Xian, X., Terrera, M., Krishnan, R., Miller, D., Bridgeman, D., et al. 2014. A pocket- sized metabolic analyzer for assessment of resting energy expenditure. Clin. Nutr. 33: 341-7. DOI: 10.1016/j.clnu.2013.06.001 Page 25 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 27 Table 1. Resting metabolic rate prediction equations. 1Units for these equations are: RMR (kcal/d), FFM (kg), BW (kg), H (cm), age (y); 2Newly-developed equations presented in this article Type of equation Reference Equation1 Organ/tissue equation Hayes (2002) 𝑅𝑀𝑅 = 𝑅𝑀𝑅𝐵 + 𝑅𝑀𝑅𝑆𝑀 + 𝑅𝑀𝑅𝑏𝑜𝑛𝑒 + 𝑅𝑀𝑅𝐴𝑇 + 𝑅𝑀𝑅𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 FFM-based equations Cunningham (1991) 𝑅𝑀𝑅 = 21.6 ∗ 𝐹𝐹𝑀 + 370 Cunningham (1980) 𝑅𝑀𝑅 = 22 ∗ 𝐹𝐹𝑀 + 500 Mifflin (1990) 𝑅𝑀𝑅 = 19.7 ∗ 𝐹𝐹𝑀 + 413 Owen (1987) M: 𝑅𝑀𝑅 = 22.3 ∗ 𝐹𝐹𝑀 + 290 F: 𝑅𝑀𝑅 = 19.7 ∗ 𝐹𝐹𝑀 + 334 ten Haaf (2014) 𝑅𝑀𝑅 = 0.239(95.272 ∗ 𝐹𝐹𝑀 + 2026.161) Tinsley2 𝑅𝑀𝑅 = 25.9 ∗ 𝐹𝐹𝑀 + 284 BW-based equations Mifflin (1990) 𝑅𝑀𝑅 = 9.99 ∗ 𝐵𝑊 + 6.25 ∗ 𝐻 ― 4.92 ∗ 𝑎𝑔𝑒 + 166 ∗ 𝑠𝑒𝑥 ― 161 Harris-Benedict (1918) M: 𝑅𝑀𝑅 = 13.75 ∗ 𝐵𝑊 + 5 ∗ 𝐻 ― 6.76 ∗ 𝑎𝑔𝑒 + 66.47 F: 𝑅𝑀𝑅 = 9.56 ∗ 𝐵𝑊 + 1.85 ∗ 𝐻 ― 4.68 ∗ 𝑎𝑔𝑒 + 655.1 FAO (1985) M: 𝑅𝑀𝑅 = 15.3 ∗ 𝐵𝑊 + 679 F: 𝑅𝑀𝑅 = 14.7 ∗ 𝐵𝑊 + 496 De Lorenzo (1999) 𝑅𝑀𝑅 = 9 ∗ 𝐵𝑊 + 11.7 ∗ 𝐻 ― 857 ten Haaf (2014) 𝑅𝑀𝑅 = 0.239(49.94 ∗ 𝐵𝑊 + 24.59 ∗ 𝐻 ― 34.014 ∗ 𝑎𝑔𝑒 + 799.257 ∗ 𝑠𝑒𝑥 + 122.502) Tinsley2 𝑅𝑀𝑅 = 24.8 ∗ 𝐵𝑊 + 10 Page 26 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 28 Table 2. Participant Characteristics Data presented as mean ± SD All (n=27) Males (n=17) Females (n=10) Age (y) 25.9 ± 6.0 26.0 ± 6.5 25.8 ± 5.4 Height (cm) 175.6 ± 9.2 180.4 ± 7.2 167.5 ± 5.7 Weight (kg) 82.9 ± 17.0 94.0 ± 9.7 63.8 ± 5.7 BMI (kg/m2) 26.6 ± 3.5 28.8 ± 2.0 22.8 ± 1.6DXA Body fat (%) 15.0 ± 4.4 12.5 ± 2.7 19.2 ± 3.4 DXA FFMI (kg/m2) 21.8 ± 3.4 24.2 ± 1.3 17.7 ± 0.9 Page 27 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 29 Table 3. Validity of resting metabolic rate estimates in males and females combined (n = 27). Method Mean ± SD p-value (t-test) CE ± SD Cohen's d r p-value (correlation) R2 SEE TE 95% LOA B p-value (linear regression) ICPARVO 2051 ± 457 --- --- --- --- --- --- --- --- --- --- --- ICCOSMED 2121 ± 482 0.037 69 ± 164 0.15t 0.94p < 0.0001* 0.88 167 175 ± 321 0.054 0.45 ICBREEZING 1753 ± 319 0.003* -298 ± 479 -0.76m 0.28s 0.16 0.08 312 557 ± 940 -0.549 0.067 OTHAYES 1896 ± 398 0.0002* -155 ± 188 -0.36s 0.91p < 0.0001* 0.83 166 241 ± 369 -0.146 0.10 FFMCUNN1 1999 ± 358 0.16 -53 ± 188 -0.13t 0.92p < 0.0001* 0.85 141 192 ± 368 -0.253 0.004† FFMCUNN2 1842 ± 352 <0.0001* -210 ± 190 -0.51s 0.92p < 0.0001* 0.85 138 281 ± 372 -0.272 0.002† FFMMIFFLIN 1755 ± 321 <0.0001* -296 ± 204 -0.75m 0.92p < 0.0001* 0.85 126 357 ± 399 -0.365 < 0.001† FFMOWEN 1778 ± 400 <0.0001* -273 ± 179 -0.64m 0.92p < 0.0001* 0.85 158 325 ± 351 -0.14 0.094 FFMT-H 2036 ± 371 0.66 -16 ± 184 -0.04t 0.92p < 0.0001* 0.85 146 181 ± 360 -0.218 0.011† BWMIFF 1741 ± 297 <0.0001* -310 ± 232 -0.80m 0.90p <0.0001* 0.80 134 385 ± 455 -0.446 <0.0001† BWH-B 1852 ± 343 <0.0001* -199 ± 209 -0.49s 0.90p <0.0001* 0.81 151 286 ± 411 -0.302 0.002† BWFAO 1848 ± 363 <0.0001* -203 ± 215 -0.49s 0.89p <0.0001* 0.79 170 293 ± 421 -0.244 0.017† BWDEL 1943 ± 252 0.04 -108 ± 259 -0.29s 0.89v <0.0001* 0.80 116 276 ± 508 -0.611 <0.0001† BWTH 1960 ± 342 0.04 -92 ± 221 -0.23s 0.89v <0.0001* 0.79 161 235 ± 432 -0.307 0.004† For RMR prediction, the following equations were used: Hayes et al. (2002), Cunningham (1980) [CUNN1], Cunningham (1991) [CUNN2], Mifflin et al. (1990), Owen et al. (1987), ten Haaf (2014) [TH], Harris and Benedict (1918) [H-B], FAO (1985) and DeLorenzo (1999) [DEL]. *statistically significant at p < 0.0033 (adjustment for multiple comparisons); †statistically significant at p < 0.05 Italic superscripts indicate magnitude of effect size or correlation (t: trivial, s: small, m: moderate, l: large, v: very large, p: near perfect). Abbreviations: B (regression coefficient), BW (body weight model), CE (constant error), F (female), FFM (fat-free mass model), IC (indirect calorimetry), LOA (limits of agreement), M (male), OT (organ/tissue model), SEE (standard error of the estimate), TE (total error). Page 28 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 30 Table 4. Validity of resting metabolic rate estimates in males (n = 17). Method Mean ± SD p-value (t-test) CE ± SD Cohen's d r p-value (correlation) R2 SEE TE 95% LOA B p-value (linear regression) ICPARVO 2337 ± 310 --- --- --- --- --- --- --- --- --- --- --- ICCOSMED 2408 ± 350 0.15 71 ± 192 0.21s 0.84v < 0.0001* 0.70 198 199 ± 376 0.134 0.396 ICBREEZING 1824 ± 359 0.0003* -513 ± 469 -1.53l 0.02t 0.93 0.00 370 686 ± 919 0.286 0.576 OTHAYES 2166 ± 199 0.005 -172 ± 217 -0.66m 0.72v 0.001* 0.51 143 272 ± 426 -0.502 0.023† FFMCUNN1 2245 ± 170 0.09 -92 ± 212 -0.37s 0.76v 0.0003* 0.57 115 226 ± 416 -0.655 0.002† FFMCUNN2 2083 ± 167 0.0002* -254 ± 213 -1.02m 0.76v 0.0003* 0.57 113 328 ± 418 -0.673 0.001† FFMMIFFLIN 1975 ± 152 < 0.0001* -362 ± 218 -1.48m 0.76v 0.0003* 0.57 103 419 ± 428 -0.763 < 0.001† FFMOWEN 2058 ± 172 0.0001* -279 ± 212 -1.11m 0.76v 0.0003* 0.57 116 346 ± 415 -0.641 0.002† FFMT-H 2290 ± 176 0.37 -47 ± 210 -0.19t 0.76v 0.0003* 0.57 119 210 ± 413 -0.620 0.003† BWMIFF 1944 ± 144 <0.0001* -393 ± 238 -1.63l 0.67l 0.003* 0.45 110 456 ± 467 -0.854 0.0005† BWH-B 2086 ± 176 0.0004* -251 ± 233 -1.00m 0.67l 0.003* 0.44 136 338 ± 457 -0.650 0.008† BWFAO 2102 ± 160 0.001* -235 ± 241 -0.95m 0.64l 0.005 0.41 128 332 ± 473 -0.758 0.003† BWDEL 2032 ± 180 <0.0001* -305 ± 224 -1.20l 0.70v 0.001* 0.49 133 374 ± 438 -0.724 0.001† BWTH 2192 ± 168 0.02 -145 ± 241 -0.58s 0.63l 0.006 0.40 135 275 ± 473 -0.709 0.006† See footnotes on Table 3. Page 29 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 31 Table 5. Validity of resting metabolic rate estimates in females (n = 10). Method Mean ± SD p-value (t-test) CE ± SD Cohen's d r p-value (correlation) R2 SEE TE 95% LOA B p-value (linear regression) ICPARVO 1566 ± 133 --- --- --- --- --- --- --- --- --- --- --- ICCOSMED 1633 ± 182 0.08 67 ± 109 0.42s 0.80l 0.003 0.65 115 124 ± 214 0.343 0.169 ICBREEZING 1633 ± 200 0.30 67 ± 194 0.4s 0.38m 0.28 0.14 196 196 ± 380 0.569 0.24 OTHAYES 1438 ± 126 0.01 -128 ± 131 -0.99m 0.49m 0.14 0.24 116 178 ± 257 -0.078 0.855 FFMCUNN1 1581 ± 107 0.68 16 ± 116 0.13t 0.55l 0.09 0.30 95 111 ± 228 -0.283 0.471 FFMCUNN2 1432 ± 105 0.005 -134 ± 116 -1.12m 0.55l 0.09 0.30 93 173 ± 227 -0.307 0.436 FFMMIFFLIN 1381 ± 96 0.0006* -184 ± 113 -1.59l 0.55l 0.09 0.30 85 213 ± 222 -0.421 0.285 FFMOWEN 1302 ± 96 < 0.0001* -263 ± 113 -2.27v 0.55l 0.09 0.30 85 284 ± 222 -0.421 0.285 FFMT-H 1604 ± 110 0.33 38 ± 117 0.31s 0.55l 0.09 0.30 98 118 ± 230 -0.240 0.542 BWMIFF 1396 ± 95 0.004 -169 ± 139 -1.47l 0.29s 0.41 0.08 96 215 ± 273 -0.519 0.332 BWH-B 1454 ± 70 0.02 -111 ± 128 -1.04m 0.33s 0.02 0.11 71 165 ± 252 -0.883 0.076 BWFAO 1417 ± 77 0.01 -149 ± 156 -1.37l -0.04t 0.92 0.00 82 210 ± 306 -1.03 0.142 BWDEL 1677 ± 107 0.04 111 ± 142 0.92m 0.31s 0.37 0.10 108 175 ± 279 -0.333 0.526 BWTH 1566 ± 112 0.99 0 ± 142 0.00t 0.26s 0.46 0.07 114 142 ± 294 -0.277 0.619 See footnotes on Table 3. Page 30 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 32 Table 6. Preliminary evaluation of new resting metabolic rate prediction equations. Method Mean ± SD p-value (t-test) CE ± SD Cohen's d r p-value (correlation) R2 SEE TE TE (LOO) 95% LOA B p-value (linear regression) All (n=27) 2051 ± 457 --- --- --- --- --- --- --- --- --- --- --- --- M (n=17) 2337 ± 310 --- --- --- --- --- --- --- --- --- --- --- ---ICPARVO F (n=10) 1566 ± 133 --- --- --- --- --- --- --- --- --- --- --- --- All (n=27) 2049 ± 422 0.93 -3 ± 176 -0.01t 0.92p < 0.0001* 0.85 166 173 187 ± 346 -0.084 0.31 M (n=17) 2338 ± 200 0.99 1 ± 205 0.00t 0.76v 0.0003* 0.57 135 199 214 ± 402 -0.484 0.018†FFMTINSLEY F (n=10) 1557 ± 126 0.83 -9 ± 123 -0.07t 0.55l 0.09 0.30 111 117 129 ± 241 -0.074 0.852 All (n=27) 2065 ± 422 0.70 13 ± 180 0.03t 0.92p <0.0001* 0.85 169 177 188 ± 352 -0.083 0.32 M (n=17) 2342 ± 241 0.92 5 ± 204 0.02t 0.75v 0.0003* 0.56 164 198 211 ± 401 -0.283 0.159BWTINSLEY F (n=10) 1593 ± 140 0.50 28 ± 137 0.20s 0.50s 0.13 0.25 129 132 140 ± 268 0.070 0.868 *statistically significant at p < 0.0033 (adjustment for multiple comparisons); †statistically significant at p < 0.05 Italic superscripts indicate magnitude of effect size or correlation (t: trivial, s: small, m: moderate, l: large, v: very large, p: near perfect). Abbreviations: B (regression coefficient), BW (body weight model), CE (constant error), F (female), FFM (fat-free mass model), IC (indirect calorimetry),LOA (limits of agreement), LOO (leave-one-out cross-validation), M (male), OT (organ/tissue model), SEE (standard error of the estimate), TE (total error). Page 31 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft 33 FIGURE LEGENDS Figure 1. Bland-Altman plots for resting metabolic rate estimation via portable indirect calorimeters and an organ/tissue equation. Results are shown for Cosmed FitmateTM (A), Breezing® (B), and the Hayes et al. (2002) equation (C). The middle horizontal line indicates the constant error (i.e. mean difference), while the upper and lower horizontal lines indicate the 95% limits of agreement. The regression line best fitting the data is shown, with larger slopes indicating greater proportional bias. Figure 2. Bland-Altman plots for resting metabolic rate estimation via body weight equations. Results are shown for Mifflin (A), Harris-Benedict (B), FAO (C), DeLorenzo (D), ten Haaf (E) and Tinsley (F). The middle horizontal line indicates the constant error (i.e. mean difference), while the upper and lower horizontal lines indicate the 95% limits of agreement. The regression line best fitting the data is shown, with larger slopes indicating greater proportional bias. Figure 3. Bland-Altman plots for resting metabolic rate estimation via fat-free mass equations. Results are shown for Cunningham (1991) (A), Cunningham (1980) (B), Mifflin (C), Owen (D), ten Haaf (E) and Tinsley (F). The middle horizontal line indicates the constant error (i.e. mean difference), while the upper and lower horizontal lines indicate the 95% limits of agreement. The regression line best fitting the data is shown, with larger slopes indicating greater proportional bias. Figure 4. Newly developed resting metabolic rate prediction equations. A strong linear relationship was observed between resting metabolic rate and body weight (A) and between resting metabolic rate and fat-free mass (B). The newly developed equations produced strong correlations with reference resting metabolic rate estimates (body weight: r = 0.921, R2 = 0.849 [C]; fat-free mass: r = 0.923, R2 = 0.851 [D]). Page 32 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 5 0 0 0 - 5 0 0 - 1 0 0 0 - 1 5 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 A B C Page 33 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 D E F A B C Page 34 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 AVERAGED RMR (kcal/d) 30002500200015001000 M E A N D IF F E R E N C E ( k c a l/ d ) 6 0 0 3 0 0 0 - 3 0 0 - 6 0 0 - 9 0 0 D E F A B C Page 35 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y. Draft BODY WEIGHT (kg) 1 1 01 0 09 08 07 06 05 0 R M R ( k c a l/ d ) 3000 2500 2000 1500 1000 FFM (kg) 1 0 09 08 07 06 05 04 0 R M R ( k c a l/ d ) 3000 2500 2000 1500 1000 R E F E R E N C E R M R ( kc al /d ) 3000 2500 2000 1500 1000 BW-PREDICTED RMR (kcal/d) 30002500200015001000 FFM-PREDICTED RMR (kcal/d) 30002500200015001000 R E F E R E N C E R M R ( kc al /d ) 3000 2500 2000 1500 1000 A B C D Page 36 of 37 https://mc06.manuscriptcentral.com/apnm-pubs Applied Physiology, Nutrition, and Metabolism A pp l. Ph ys io l. N ut r. M et ab . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y St . F ra nc is X U ni v on 0 9/ 22 /1 8. F or p er so na l u se o nl y.
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