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

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Resting Energy Expenditure, Metabolic and Sex Hormones in Two Phases of the Menstrual and Hormonal Contraceptive Cycles © 2024 American College of Sports Medicine Accepted version (Final draft) Löfberg, Ida E.; Karppinen, Jari E.; Laatikainen-Raussi, Vesa; Lehti, Maarit; Hackney, Anthony C.; Ihalainen, Johanna K.; Mikkonen, Ritva S. Löfberg, I. E., Karppinen, J. E., Laatikainen-Raussi, V., Lehti, M., Hackney, A. C., Ihalainen, J. K., & Mikkonen, R. S. (2024). Resting Energy Expenditure, Metabolic and Sex Hormones in Two Phases of the Menstrual and Hormonal Contraceptive Cycles. Medicine and Science in Sports and Exercise, ahead of Print . https://doi.org/10.1249/mss.0000000000003518 2024 Published ahead of Print Medicine & Science in Sports & Exercise® Published ahead of Print contains articles in unedited manuscript form that have been peer reviewed and accepted for publication. This manuscript will undergo copyediting, page composition, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered that could affect the content. Copyright © 2024 American College of Sports Medicine Resting Energy Expenditure, Metabolic and Sex Hormones in Two Phases of the Menstrual and Hormonal Contraceptive Cycles Ida E. Löfberg1, Jari E. Karppinen1,2, Vesa Laatikainen-Raussi1, Maarit Lehti1, Anthony C. Hackney3, Johanna K. Ihalainen1,4, and Ritva S. Mikkonen5 1Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, FINLAND; 2Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, FINLAND; 3 Department of Exercise & Sport Science – Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC; 4Finnish Institute of High Performance Sport KIHU, Jyväskylä, FINLAND; 5Sports Technology Unit, Faculty Accepted for Publication: 9 July 2024 ACCEPTED Downloaded from http://journals.lww.com/acsm-msse by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCyw CX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 08/09/2024 Resting Energy Expenditure, Metabolic and Sex Hormones in Two Phases of the Menstrual and Hormonal Contraceptive Cycles Ida E. Löfberg1, Jari E. Karppinen1,2, Vesa Laatikainen-Raussi1, Maarit Lehti1, Anthony C. Hackney3, Johanna K. Ihalainen1,4, and Ritva S. Mikkonen5 1Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, FINLAND; 2Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, University of Helsinki, Helsinki, FINLAND; 3 Department of Exercise & Sport Science – Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC; 4Finnish Institute of High Performance Sport KIHU, Jyväskylä, FINLAND; 5Sports Technology Unit, Faculty of Sport and Health Sciences, University of Jyväskylä, Vuokatti, FINLAND Address for correspondence: Ida Löfberg, Faculty of Sport and Health Sciences, PO Box 35. FI-40014 University of Jyväskylä, Jyväskylä, Finland; E-mail: [email protected]. Conflict of Interest and Funding Source: The data presented here are part of a larger Women’s menstrual cycle and endurance training (NaisQs) study. The study was funded by the Finnish Ministry of Education and Culture and Firstbeat Analytics Expense funding for blood analyses was received from the Suomen Urheilututkimussäätiö. The authors declare no conflicts of interest, financial or otherwise, regarding this study. Medicine & Science in Sports & Exercise, Publish Ahead of Print DOI: 10.1249/MSS.0000000000003518 ACCEPTED This is an open-access article distributed under the terms of the Creative Commons AttributionNon Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. ACCEPTED ABSTRACT -luteal phases of the MC in NoOC-group (n=38) or the active and inactive phases of the COC cycle (COC, n=19). Participants recorded their food intake for 3 days after measurements. A secondary analysis was completed for the NoOC-group without REE outliers (difference between measurements >1.5 × interquartile range, n=4). Results: In the NoOC-group, luteal phase REE was 40 kcal higher than follicular phase REE [95% confidence interval (CI): -2 kcal/d–82 kcal/d, d=0.20, p=0.061]. Leptin (d=0.35, p<0.001), T3 (d=0.26, p=0.05) and fat intake (d=0.48, p=0.027) were lower, and T4 (d=0.21, p=0.041) was higher in the luteal phase. After excluding outliers, REE was 44 kcal higher in the luteal phase than in the follicular phase (95% CI: 12kcal/d–76kcal/d, d=0.22, p=0.007). In the COC-group, the mean difference in REE was -2 kcal (95% CI-82 kcal/d–79 kcal/d) between active and inactive phases, while T3 was higher in the inactive phase (d=0.01, p=0.037). Conclusions: REE increases only slightly from the follicular to the luteal phase but remains unchanged between COC phases. Increases in T3, leptin, and fat intake during the luteal phase might echo metabolic fluctuations that parallel female sex hormones during the MC. ACCEPTED Key Words: FEMALE PHYSIOLOGY, ENERGY INTAKE, HORMONAL CONTRACEPTION, REPRODUCTIVE HORMONES, RESTING METABOLISM ACCEPTED INTRODUCTION The menstrual cycle (MC) is a series of finely tuned physiological processes caused by fluctuations in endogenous sex hormones. These hormonal fluctuations cause changes not only in the structure and function of the ovaries but also influence various metabolic pathways that regulate energy metabolism (1). Resting energy expenditure (REE) typically accounts for 60 to 75% of total daily energy expenditure and is reflective primarily of fat-free mass (FFM) and fat mass (FM), which are associated with functional demands of organs and tissues (2). Nevertheless, the possible influence of sex hormones on REE remains equivocal and requires more attention to improve our understanding of female physiology (3). There is some evidence that REE increases in the luteal phase of the MC compared to the follicular phase. High levels of progesterone (P4) accompanied by estradiol (E2) during the mid-luteal phase are proposed to contribute to REE through their thermoregulatory interactions (4,5). In a recent systematic review (3), 47% of 26 studies reported higher REE in the luteal phase, although the varying quality of the studies limited the interpretation of the results. Of particular concern was the inconsistency or lack of MC phase verification in included studies as it has been proposed that the MC should be divided into four distinct phases according to hormonal profiles when assessing the influence of sex hormones and their ratios on metabolism (3,6). In addition to sex hormones, habitual food intake may increase during the luteal phase compared to the follicular phase (7). Regrettably, not all previous research has been able to account for habitual food intake, a variable of significance when studying the relationship ACCEPTED between the menstrual cycle and metabolism. Considering that food intake is closely related to REE (8), MC-dependent patterns mediating the effects of food intake on REE should not be ignored. Sex hormones E2 and P4 may interact with metabolic hormones that are involved in the regulation of REE and food intake. For example, thyroid hormones play an important role in energy metabolism by regulating complex cell functions and contributing to thermogenesis and therefore, stimulating energy expenditure (9,10). Previous studies have not, however, consistently showed that thyroid hormone levels fluctuate during the MC (11). Adipocytederived leptin and the gut hormone ghrelin are considered appetite-regulating hormones but are also strongly associated with energy metabolism (12). Leptin is known to be involved in the regulation of the MC through the link between energy status and reproduction and its increase during the luteal phase have been consistently reported (13) while ghrelin does not appear to be influenced by the MC (14). There is, however, evidence that leptin does not directly influence the processes underlying REE (15) whereas ghrelin is shown to be negatively associated with REE independent of body fat (16). The synthetic female sex hormones contained in monophasic combined oral contraceptives (COC) downregulate endogenous E2 and P4 and appear to only trivially affect other physiological processes (17). Regrettably, the interaction of COC with REE is not clear, but a recent review suggests that COC use does not affect food intake (18). Studies investigating the effects of COC phases on metabolic hormones are scarce, but currently there appears to be ACCEPTED consensus on unchanged thyroid hormones (19), leptin (20) and ghrelin (21) levels during the COC cycle. Considering that a woman has an average of 450 MCs during her lifetime (22), regular fluctuation in energy metabolism can have meaningful effects on her health. Moreover, according to United Nations (23), approximately 16% (151 million) of women use COC, which underlines the importance of further research on the metabolic effects of hormonal contraception use. As such, the aim of this study was to investigate the effects of endogenous and exogenous female sex hormones on REE and metabolic hormones in naturally menstruating women and women using COC. We hypothesized that REE, energy intake, and leptin would be higher in the luteal phase than in the follicular phase, while no changes would occur between COC cycle phases. MATERIAL AND METHODS Participants Healthy untrained women (n = 77, Tier 1: recreationally active, defined according to the Participant Classification Framework (24)) aged 18-35 years with a self-reported body mass index (BMI) between 19.5 and 35 kg/m2 at recruitment volunteered to participate in this study via the advertisement in social media, sport halls, gyms, public places, and university mailing lists. The participants were stratified into two groups, one not using any hormonal contraception with a self-reported regular 26–35-day MC over the previous six months (NoOCgroup, n=58, normally menstruating), and one group using monophasic COC (COC-group, ACCEPTED Statistical analysis Statistical analyses were conducted using SPSS Statistics 28 (SPSS Inc., Chicago, IL) and figures were graphed with GraphPad Prism. Results are reported as mean ± SD or as medians with first and third quartiles. The effect size for results is expressed as Cohen’s d (32). The normality of the data was tested by Shapiro-Wilk. Within-group changes were analyzed by Student’s paired t-test for normally distributed data and Wilcoxon’s signed-rank test for nonnormally distributed data. Associations between variables of interest and REE were tested by multiple regression analysis using FFM and FM as covariates per recommendations (2). Consistency within resting metabolism measures were assessed using the intraclass correlation coefficient (ICC) with a one-way random-effects model (Supplemental Table 2, Supplemental Digital Content, ICCof REE and RER between two measurements in NoOCand COC- -determined level of statistical significance was p≤0.05. Inspection of the data revealed four participants in the NoOC group as potential outliers because their REE change from the follicular to the luteal phase exceeded 1.5 × the interquartile range (33). Although we found no physiological or measurement-related reasons for these observations, outliers have the potential to excessively influence the results in parametric tests compared with other observations. Therefore, a secondary analysis was conducted, excluding these four participants when using REE as the outcome. RESULTS The physical characteristics of participants are presented in Table 1. Results for the NoOCand COC-groups are presented separately because the groups are hormonally different, ACCEPTED and the primary purpose of this investigation was to observe within-group changes in two homogeneous samples. Differences between follicular and luteal phases in the NoOC-group Table 2 shows the hormone concentrations, resting metabolism, body composition, and dietary measures during the follicular and luteal phases. E2 concentrations were lower in the follicular phase compared to the luteal phase (p<0.001, d=1.83) whereas all participants demonstrated P4 values > 16 nmol/l in the luteal phase, indicating expectedly higher P4 concentrations compared to the follicular phase (p<0.001, d=4.79). Leptin (p<0.001, d=0.35) and T3 (p=0.05, d=0.26) were lower in the follicular phase compared to the luteal phase, while T4 was higher (p=0.042, d=0.21) in the follicular phase (Figure 1A–C). In the full sample, REE data were compatible with a -2 kcal/d decrease to 82 kcal/d increase (95% CI) from the follicular to the luteal phase (mean difference 40 kcal/d, Figure 2A). Additionally, RER remained similar between MC phases. However, the secondary analysis indicated significantly higher REE in the luteal phase compared to the follicular phase (mean difference 44 kcal/d, 95% CI: 13 kcal/d to 76 kcal/d, p=0.007) (Figure 2B). We observed a strong ICC for REE in both the full sample and after excluding outliers (p<0.001), whereas RER showed a weak ICC in both cases (p=0.451 and p=0.311, respectively) (Supplemental Table 2, Supplemental Digital Content). Fat intake was found to be higher during the luteal phase compared to the follicular phase (mean difference 13 g/d, 95% CI: 2 g/d to 24 g/d, p=0.027) (Figure 1D). The change in ACCEPTED energy intake was not statistically significant, but results favored higher energy intake during the luteal phase, with a mean difference of 147 kcal/d (95% CI: -5 kcal/d to 300 kcal/d, p=0.058). Associations of hormones and dietary measures with FFMand FM-adjusted REE in the NoOC-group The associations of hormone concentrations and dietary measures with REE adjusted for FFM and FM are shown in Table 3. FFM and FM explained 48% and 58% of the variance of REE in the follicular and luteal phases, respectively. T3 was positively associated with REE in both phases, increasing the adjusted R2 to 51% in the follicular phase and 69% in the luteal phase. Of the dietary measures, energy intake (adjusted R2=55%), energy availability (adjusted R2=50%), fat intake (adjusted R2=49%), and carbohydrate intake (adjusted R2=51%) were positively associated with REE in the follicular phase but not in the luteal phase. Secondary analysis without REE outliers indicated that the ability of FFM and FM to explain REE variability improved (adjusted R2 55% and 64% in the follicular and luteal phases, respectively). The reported associations between T3 and dietary measures, and REE remained similar but were stronger after exclusion of outliers (Supplemental Table 3, Supplemental Digital Content, The associations of hormone concentrations and dietary measures with resting energy expenditure, adjusted for FFM and FM, at FOL and LUT phases). T3 increased the adjusted R2 to 64% (β=0.35, p=0.002) in the follicular phase and 75% (β=0.38, p<0.001) in the luteal phase. Associations between REE and energy intake (adjusted R2=67%, β=0.49, p<0.001), energy availability (adjusted R2 =60%, β=0.42, p<0.005), fat intake (adjusted ACCEPTED R2=58%, β=0.37, p<0.010), and carbohydrate intake (adjusted R2=60%, β=0.40, p<0.005), were found in follicular phase. Differences between active and inactive phases in the COC-group The hormone concentrations, body composition, resting metabolism and dietary measures in the COC-group are presented in Table 4. T3 (p<0.001, d=0.93) (Figure 3A) and body mass (p=0.037, d=0.058) were higher in the inactive phase compared to the active phase, but T4 and leptin did not change from the active phase to the inactive phase (Figure 3B–C). The mean difference for REE change between phases was -2 kcal (95% CI: -82 kcal/d to 79 kcal/d, p=0.969) (Figure 3D) and for fat intake -1 g (95% CI: 17 g/d to 15 g/d, p=0.885) (Figure 3E). The ICC for both REE (p=0.005) and RER (p=0.027) was strong (Supplemental Table 2, Supplemental Digital Content). Associations of hormones and dietary measures with FFMand FM-adjusted REE in the COC-group Table 5 displays associations between hormone concentrations and dietary measures with REE adjusted for FFM and FM. FFM and FM together explained 37% and 66% of the variance in REE in the active and inactive phases, respectively. Leptin was negatively associated with REE in the inactive phase, increasing the adjusted R2 to 71%, and unAG was negatively associated with REE in the active phase (adjusted R2=50%). Other hormones or dietary measures did not appear to contribute to REE along with FFM and FM in either phase. ACCEPTED DISCUSSION The present study investigated REE and metabolic hormones over one menstrual or COC cycle, to our knowledge, in the largest sample to date. Our findings suggested higher REE during the luteal phase compared to the follicular phase (Figure 2), while no notable REE differences between COC cycle phases were observed (Figure 3D). In the NoOC-group, concentrations of T3, leptin, and fat intake were higher in the luteal phase compared to the follicular phase (Figure 1). Conversely, in the COC-group, only T3 and body mass were significantly higher during the inactive phase compared to the active phase. These findings underscore the intricate relationship between hormonal fluctuations and resting metabolism, emphasizing the necessity to consider menstrual cycle phase when interpreting REE measurements. Metabolism and energy intake in the follicular and luteal phases Our results suggested that REE was approximately 40 kcal/d higher in the luteal phase than in the follicular phase; however, the difference became statistically significant only after excluding four outliers who influenced the results (Figure 2B). Of these outliers, two had higher REE in the follicular phase, and the other two had higher REE in the luteal phase. Importantly, their exclusion improved our ability to explain REE variation using FFM and FM, justifying the value of our secondary analysis. However, despite investigating the largest sample to date, our study may still be underpowered to draw precise estimates of the MC effect on REE. The intraindividual day-to-day variation in REE has been reported to be approximately 5% (34), consistent with the test-retest reliability observed in our laboratory. This indicates that the daily ACCEPTED variation in REE may be even larger than the effect of the MC. Therefore, we recommend that our results be interpreted in the light of previous literature. Nevertheless, our findings are consistent with previous systematic review and meta-analysis by Benton et al. (3), who reported a slightly higher (d=0.33, p<0.001) REE in the luteal phase compared with the follicular phase. However, particularly the inclusion of studies published in year 2000 or after led to very similar effect size to ours (d=0.23, p=0.055). Furthermore, whether the more methodologically comparable previous studies have reported REE differences classified as statistically significant (35) or not (36), the numerical differences between early follicular and luteal phase tend to fall into the same range as observed in our study. For instance, Day et al. (35) reported a 29 kcal higher REE in the luteal phase, while Elliott et al. (36) observed variations ranging from 8 kcal to 57 kcal higher REE in the luteal phase compared to the early follicular phase across two MCs. Therefore, taking into consideration the totality of current evidence, the effect of MC on REE seems probable. Whether the effect has profound physiological significance remains uncertain. Our findings suggest that increase of REE could be driven by T3, which is well known to stimulate energy expenditure (37). In our study, T3 levels showed MC-related fluctuations that paralleled REE and were associated with FFMand FM-adjusted REE during both MC phases (Table 3). However, the MC-associated T3 response has been previously reported only by Lariviere et al. (38), while other studies have found more stable T3 levels during the MC (21,11,39). This discrepancy could be attributed to the functional nature of thyroid hormones (40), coupled with methodological differences such as variations in sample ACCEPTED timing and assay techniques across studies. We also noted higher leptin concentrations in the luteal phase (Figure 1C), consistent with previous research (13,21,41). However, leptin levels were not associated with REE, which agrees with previous research (15). Of other potential metabolic hormones, we found stable levels of UnAG and AG throughout the MC. Moreover, their levels were not associated with REE, indicating that these hormones are unaffected by MC and are unlikely to explain MC-associated changes in REE. Nevertheless, further investigation is required to elucidate the complex interplay between female sex hormones, other metabolic hormones, and energy expenditure. Considering energy intake as a part of the energy balance equation, we observed higher fat intake during the luteal phase compared to the follicular phase (Figure 1D), which is in line with few previous studies (42, 43). For energy intake, our data indicated it to remain constant or increase from the follicular to the luteal phase, of which the latter agrees with the widely held view that energy intake is higher during the luteal phase compared to the late follicular phase (7,44–46). The hypothesized physiological reason for these variations is that E2 may inhibit appetite in the late follicular phase (47) via a complex process involving hypothalamic E2 neurons and their regulatory role in energy homeostasis (48). Conversely, P4 is suggested to stimulate appetite in the presence of E2 during the luteal phase (47). These changes could also be a response to the possible increase in REE to maintain energy balance (49). However, an increase in energy intake during the luteal phase has not consistently been reported (21,50). It should be emphasized that the control of food intake is influenced by psychological and social factors as well as physiological mechanisms (51), which underlines the uncertainty of evaluating energy intake data. ACCEPTED In this study, all dietary measures, except protein intake, contributed to the FFMand FM-adjusted REE in the follicular phase (Table 3). In the long-term, energy intake naturally reflects body size, which explains the relationship between REE and energy intake (8,52). Diet prior to REE measurement is known to influence the results through the increased thermic effect of food (26). However, herein dietary intake was recorded for 3 days after the REE measurement for practical reasons. Nevertheless, the stable RER observed between the follicular and luteal phase indicates a lack of dramatic changes in energy balance and substrate metabolism preceding the measurement (53), which is also supported by the lack of significant changes in energy intake according to the food diaries in this study. Additionally, REE measurement was conducted in a fasted state to mitigate thermic effect of food. The underlying reason for the associations observed only during the follicular phase, but not luteal phase, is unknown. However, it can be speculated that the energy intake replaced some of the mass-related aspects of REE in the follicular phase. This speculation is supported by our finding that FFM and FM explained a smaller proportion of REE variability in the follicular phase than in the luteal phase (48% vs. 58%). Furthermore, adding energy intake to the model increased the explanatory percentage from 48% to 59% in the follicular phase, similar to the explanatory percentage of FFM and FM (58%) in the luteal phase. This observation implies that energy intake might play a more significant role in REE variability in the follicular phase, providing insight into the complex nature of energy metabolism in women and indicating the need for further investigation. ACCEPTED Metabolism and energy intake in the active and inactive phases This study may be the first to examine REE differences between the active and inactive phase of the COC cycle while also reporting hormonal concentrations. In the present study, both endogenous E2 and P4 remained stable during the COC cycle as expected. Our results indicate no evidence of a difference in REE between COC phases (Figure 3), which agrees with previous studies (54,55). In the present study, only T3 was higher during the inactive phase of the COC cycle (Figure 3A), which contradicts with previous studies that reported stable thyroid hormone levels throughout the COC cycle (19,21). This unexpected finding indicates that further investigation of potential factors contributing this variation in thyroid hormone levels during the COC cycle might be required. However, it is noteworthy that higher T3, T4, and TSH levels have been reported in COC-users compared to non-users (19) as well as at commencement of COC use (56,57). This difference is thought to be related to the increase of thyroxine-binding globulin caused by COCs (56). Nevertheless, higher T3 in the inactive phase of the COC cycle might be attributed to individual variations in thyroid metabolism or other unexplained factors. - and FM-adjusted REE was not associated with T3 at either phase, but leptin was inversely correlated with REE in the inactive phase while unAG had a negative correlation with REE in the active phase (Table 5). The inability of T3 to explain REE variation in the COCgroup raises questions about the influence of exogenous female sex hormones on thyroid hormone metabolism. Furthermore, the underlying physiological mechanisms driving these observed negative relationships between REE and leptin, as well as REE and UnAG, remain ACCEPTED inconclusive and warrant further investigation. Understanding these associations may serve as a means to optimize metabolic health of women using COCs. Given that only few studies have addressed within-cycle changes in body composition and dietary intake among COC users, our study is a valuable contribution to this area of research. We observed higher body mass during the inactive phase, while FM, FFM, and dietary intake remained unchanged. Some previous studies have reported that body mass and body composition do not change during the COC cycle (58,59). However, our results align with Ihalainen et al. (21) who found higher body mass during the inactive phase, suggesting that COC use might have a role in body weight fluctuations. Indeed, E2 increases plasma volume, whereas P4 is also known to influence sodium and water regulation (60). Studies examining dietary intake across the COC phases have consistently reported no changes (21,54,61), which is supported by our findings. 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Effects of an oral contraceptive containing 30 mcg ethinyl estradiol and 2 mg dienogest on thyroid hormones and androgen parameters: conventional vs. extended-cycle use. Contraception. 2008;77(6):420-5. 58. Vaiksaar S, Jürimäe J, Mäestu J, et al. No effect of menstrual cycle phase on fuel oxidation during exercise in rowers. Eur J Appl Physiol. 2011;111(6):1027-34. 59. Rael B, Alfaro-Magallanes VM, Romero-Parra N, et al. Menstrual cycle phases influence on cardiorespiratory response to exercise in endurance-trained females. Int J Environ Res Public Health. 2021;18(3):860. 60. Stachenfeld NS. Sex hormone effects on body fluid regulation. Exerc Sport Sci Rev. 2008;36(3):152–9. 61. Tucci SA, Murphy LE, Boyland EJ, Dye L, Halford JC. Oral contraceptive effects on food choice during the follicular and luteal phases of the menstrual cycle. A laboratory based study. Appetite. 2010;55(3):388-92. 62. Burrows M, Peters CE. The influence of oral contraceptives on athletic performance in female athletes. Sports Med. 2007;37(7):557–74. 63. Miles-Chan JL, Dulloo AG, Schutz Y. Fasting substrate oxidation at rest assessed by indirect calorimetry: is prior dietary macronutrient level and composition a confounder? Int J Obes (Lond). 2015;39(7):1114-7. ACCEPTED FIGURE LEGENDS Figure 1. Changes in T3 (A), n=38, T4 (B), n=38, leptin (C), n=38, and fat intake (D), n=29, at follicular phase (FOL) and luteal phase (LUT). Data are presented as means with standard deviation and individual data points. NoOC. *p≤0.05, ***p<0.001 Figure 2. Changes in REE in the full sample (A), n=38, and after the secondary analysis (B), n=34, at follicular phase (FOL) and luteal phase (LUT). Data are presented as means with standard deviation and individual data points. NoOC. **p≤0.01 Figure 3. Changes in T3 (A), n=19, T4 (B), n=19, leptin (C), n=19, REE (D), n=19, and fat intake (E), n=16, at active phase (ACT) and inactive phase (INACT). Data are presented as means with standard deviation and individual data points. COC. ***p<0.01 ACCEPTED SUPPLEMENTAL DIGITAL CONTENT ACCEPTED ACCEPTED ACCEPTED ACCEPTED Table 1. Mean ± SD physical characteristics of the NoOC- (n=38) and COC-group (n=19). NoOC-group COC-group Age (yr) 29.8 ± 3.9 26.1±4.0 Height (m) 1.66 ± 0.06 1.69 ± 0.05 Body mass (kg) 68.3 ± 10.9 68.5 ± 6.7 BMI (kg·m−2) 24.9 ± 4.0 24.1 ± 2.1 Body fat (%) 28.3 ± 8.3 27.7 ± 6.3 Length of MC or COC cycle (days) 27 ± 2 21–24 active + 4–7 inactive NoOC: naturally menstruating; COC: combined oral contraceptive; BMI: body mass index; COC: combined oral contraceptive. Significant differences bolded (p≤0.05). ACCEPTED Supplemental Digital Content 1 Supplemental Table 1. Content of COC pills and brand names used by participants in the COC-group. Brand name Progestogen component (generation) N Gestinyl Ethinylestradiol 20 µg Gestoden 75 µg (3rd) 3 Gestinyl Ethinylestradiol 30 µg Gestoden 75 µg (3rd) 2 Microgynon Ethinylestradiol 0,15 mg Levonorgestrel 30 µg (2nd) 1 Tasminetta/Yasmin Ethinylestradiol 0,03 mg Drospirenone 3 mg (4th) 2 Daisynelle Ethinylestradiol 20 µg Desogestrel 150 µg (3rd) 1 Dienorette Ethinylestradiol 0,03 mg Dienogest 2 mg (4th) 5 Yasminelle/Dizminelle/Stefaminelle Ethinylestradiol 0,02 mg Drospirenone 3 mg (4th) 3 Mercilon Ethinylestradiol 20 µg Desogestrel 150 µg (3rd) 1 Levesia Ethinylestradiol 20 µg Levonorgestrel 100 µg (2nd) 1 COC: combined oral contraceptive ACCEPTED Supplemental Figure 1. Supplemental Figure 1. Flowchart of participant enrollment through the study. FOL: follicular phase; ACT: active phase; LUT: luteal phase; INACT: inactive phase; P4: progesterone; MC: menstrual cycle; TSH: thyroid-stimulating hormone. ACCEPTED Supplemental Table 2. Intraclass correlation coefficients (ICC) of REE and RER between two measurements in NoOCand COC-groups. Results of the secondary analysis in italics. Variable Group ICC (95%CI) P-value REE NoOC 0.88 (0.76–0.94) 0.94 (0.87–0.97) <0.001*** <0.001*** COC 0.71 (0.26–0.89) 0.005* RER NoOC 0.04 (-0.84–0.50) 0.16 (-0.68–0.58) 0.451 0.311 COC 0.60 (-0.01–0.85) 0.027* REE: resting energy expenditure; RER: respiratory exchange ratio. *p≤0.05 ***p<0.001. Statistically significant p-values are bolded. ACCEPTED Supplemental Table 3. The associations of hormone concentrations and dietary measures with resting energy expenditure, adjusted for fat-free mass (FFM) and fat mass (FM), at follicular (FOL) and luteal (LUT) phases. Results of the secondary analysis. NoOC. FOL LUT B 95% CI β P-value n B 95% CI β P-value n Hormones E2 (nmol/l) 0.04 -0.21 to 0.28 0.04 0.757 34 -0.07 -0.35 to 0.21 -0.07 0.608 34 P4 (nmol/l) 22.12 -62.55 to 106.78 0.07 0.598 34 0.04 -0.21 to 0.28 0.04 0.757 34 T3 (pmol/l) 81.99 31.58 to 132.41 0.35 0.002 34 95.56 50.16 to 140.96 0.38 <0.001 34 T4 (pmol/l) 22.31 -12.52 to 57.14 0.17 0.201 34 1.88 -23.78 to 27.53 0.018 0.882 34 TSH (mIU/l) -14.98 -74.38 to 44.43 -0.06 0.610 34 39.22 -7.11 to 85.56 0.18 0.094 34 Leptin (ng/ml) 3.32 -4.84 to 11.47 0.19 0.413 34 2.42 -4.31 to 9.15 0.18 0.468 34 unAG (pg/ml) 0.12 -0.16 to 0.41 0.11 0.387 33 0.14 -0.07 to 0.36 0.15 0.182 34 AG (pg/ml) 0.11 -0.21 to 0.42 0.09 0.494 33 0.08 -0.15 to 0.31 0.08 0.486 34 Dietary measures EI (kcal·day−1) 0.21 0.10 to 0.32 0.49 <0.001 29 0.08 -0.03 to 0.18 0.22 0.133 27 EA (kcal·day−1) 0.21 0.07 to 0.35 0.42 0.005 27 0.08 -0.03 to 0.19 0.22 0.132 26 FAT (g·day−1) 2.52 0.65 to 4.39 0.37 0.010 29 1.89 -0.12 to 3.90 0.29 0.065 27 CHO (g·day−1) 1.30 0.43 to 2.17 0.40 0.005 29 0.34 -0.46 to 1.14 0.12 0.394 27 PROT (g·day−1) 0.53 -1.43 to 2.50 0.08 0.582 29 0.41 -1.64 to 2.46 0.07 0.681 27 P-values <0.05 in bold. B: unstandardized coefficient; CI: confidence interval; β: standardized coefficient; E2: estradiol; P4: progesterone; T3: triiodothyronine; T4: thyroxine; TSH: thyroid-stimulating hormone; unAG; unacylated ghrelin; AG: acylated ghrelin; EI: energy intake, EA: energy availability; FAT: fat intake; CHO: carbohydrate intake; PROT: protein intake. ACCEPTED