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Phenotyping in clinical nutrition

Skeletal muscle mass can be estimated by creatine (methyl‐d3) dilution and is correlated with fat-free mass in active young males



Assessing whole-body skeletal muscle mass (SMM) and fat-free mass (FFM) is essential for the adequate nutritional management and training evaluation of athletes and trained individuals. This study aimed to determine the relationship between SMM assessed using the creatine (methyl‐d3) dilution (D3-creatine) method and SMM estimated by whole-body magnetic resonance imaging (MRI) in healthy young men undergoing exercise training. Additionally, we examined the association between FFM measured using the four-component (4C) method (FFM4C) and the total body protein value estimated using 4C (TBpro4C).

Methods and results

We analyzed the data of 29 males (mean age, 19.9 ± 1.8 years) who exercised regularly. SMM measurements were obtained using the D3-creatine method (SMMD3-creatine) and MRI (SMMMRI). The SMMD3-creatine adjusted to 4.3 g/SMM kg was significantly higher than SMMMRI (p < 0.01). The fit of the creatine pool size compared with SMMMRI was 5.0 g/SMMMRI kg. SMMMRI was significantly correlated with both SMMD3-creatine adjusted to 4.3 g/kg and 5.1 g/kg. TBpro4C was significantly lower than SMMMRI (p < 0.01). Contrastingly, FFM4C was significantly higher than SMMMRI (p < 0.01).


SMMD3-creatine adjusted to 4.3 g/SMM kg—a previously reported value—may differ for athletes and active young males. We believe that a value of 5.0–5.1 g/SMM kg better estimates the total muscle mass in this population. Traditional FFM estimation highly correlates with SMMMRI in well-trained young males, and the relationships appear strong enough for total body protein or SMM to be estimated through the FFM value.

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Fig. 1: Whole-body skeletal muscle mass, total body protein and fat-free mass.
Fig. 2: Relative whole-body components compared to body mass and FFM.
Fig. 3: Comparison of the D3-creatine method and MRI results after the creatine pool size was adjusted to (A) 4.3 g/SMM kg and (B) 5.1 g/SMM kg.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


  1. Evans WJ, Hellerstein M, Orwoll E, Cummings S, Cawthon PM. D3 -Creatine dilution and the importance of accuracy in the assessment of skeletal muscle mass. J Cachexia Sarcopenia Muscle. 2019;10:14–21.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Balsom PD, Söderlund K, Ekblom B. Creatine in humans with special reference to creatine supplementation. Sport Med. 1994;18:268–80.

    Article  CAS  Google Scholar 

  3. Schaap LA. D3-creatine dilution to assess muscle mass. J Gerontol A Biol Sci Med Sci. 2019;74:842–3.

    Article  PubMed  Google Scholar 

  4. Cawthon PM, Orwoll ES, Peters KE, Ensrud KE, Cauley JA, Kado DM, et al. Strong relation between muscle mass determined by d3-creatine dilution, physical performance, and incidence of falls and mobility limitations in a prospective cohort of older men. J Gerontol - Ser A Biol Sci Med Sci. 2019;74:844–52.

    Article  Google Scholar 

  5. Evans WJ, Scottoline B, Imam F, Hellerstein M, Garton K, Czerwieniec G, et al. D3-creatine dilution for the noninvasive measurement of skeletal muscle mass in premature infants. Pediatr Res. 2021;89:1508–14.

    Article  CAS  PubMed  Google Scholar 

  6. Evans WJ, Shankaran M, Smith EC, Morris C, Nyangau E, Bizieff A, et al. Profoundly lower muscle mass and rate of contractile protein synthesis in boys with Duchenne muscular dystrophy. J Physiol. 2021;599:5215–27.

    Article  CAS  PubMed  Google Scholar 

  7. Orwoll ES, Peters KE, Hellerstein M, Cummings SR, Evans WJ, Cawthon PM. The importance of muscle versus fat mass in sarcopenic obesity: a re-evaluation using D3-creatine muscle mass versus DXA lean mass measurements. J Gerontol A Biol Sci Med Sci. 2020;75:1362–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rogers-soeder TS, Peters KE, Lane NE, Shikany JM, Judd S, Langsetmo L, et al. Dietary intake, D3Cr muscle mass, and appendicular lean mass in a cohort of older men. J Gerontol Ser A. 2019;75:1353–61.

    Article  Google Scholar 

  9. Ribeiro AS, Avelar A, Kassiano W, Nunes JP, Schoenfeld BJ, Aguiar AF, et al. Creatine supplementation does not influence the ratio between intracellular water and skeletal muscle mass in resistance-trained men. Int J Sport Nutr Exerc Metab. 2020;11:1–7.

    Article  CAS  Google Scholar 

  10. Clark RV, Walker AC, Connor-semmes RLO, Leonard MS, Miller RR, Stimpson SA, et al. Total body skeletal muscle mass: estimation by creatine (methyl-d3) dilution in humans. J Appl Physiol. 2014;116:1605–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Clark RV, Walker AC, Miller RR, O’Connor-Semmes RL, Ravussin E, Cefalu WT, et al. Creatine (methyl-d3) dilution in urine for estimation of total body skeletal muscle mass: Accuracy and variability vs. MRI and DXA. J Appl Physiol. 2018;124:1–9.

    Article  CAS  PubMed  Google Scholar 

  12. Weber MA, Krakowski-Roosen H, Schröder L, Kinscherf R, Krix M, Kopp-Schneider A, et al. Morphology, metabolism, microcirculation, and strength of skeletal muscles in cancer-related cachexia. Acta Oncol. 2009;48:116–24.

    Article  PubMed  Google Scholar 

  13. Willer B, Stucki G, Hoppeler H, Brühlmann P, Krähenbühl S. Effects of creatine supplementation on muscle weakness in patients with rheumatoid arthritis. Rheumatology 2000;39:293–8.

    Article  CAS  PubMed  Google Scholar 

  14. Brault JJ, Terjung RL. Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types. Am J Physiol-Cell Physiol 2003;284:1481–9.

    Article  Google Scholar 

  15. Morris-Paterson TE, Stimpson SA, Miller RR, Barton ME, Leonard MS, Carmichael O, et al. Total body skeletal muscle mass estimated by magnetic resonance imaging and creatine (methyl-d3) dilution in athletes. Scand J Med Sci Sport. 2020;30:421–8.

    Article  Google Scholar 

  16. Harris RC, Söderlund K, Hultman E. Elevation of creatine in resting and exercised muscle of normal subjects by creatine supplementation. Clin Sci. 1992;83:367–74.

    Article  CAS  Google Scholar 

  17. Prior BM, Modlesky CM, Evans EM, Sloniger MA, Saunders MJ, Lewis RD, et al. Muscularity and the density of the fat-free mass in athletes. J Appl Physiol. 2001;90:1523–31.

    Article  CAS  PubMed  Google Scholar 

  18. Jensen B, Braun W, Geisler C, Both M, Klückmann K, Müller MJ, et al. Limitations of fat-free mass for the assessment of muscle mass in obesity. Obes Facts. 2019;12:307–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sagayama H, Yamada Y, Tanabe Y, Kondo E, Ohnishi T, Takahashi H. Validation of skeletal muscle mass estimation equations in active young adults: A preliminary study. Scand J Med Sci Sport. 2021;31:1897–907.

    Article  Google Scholar 

  20. McKay AKA, Stellingwerff T, Smith ES, Martin DT, Mujika I, Goosey-Tolfrey VL, et al. Defining training and performance caliber: a participant classification framework. Int J Sports Physiol Perform. 2022;17:317–31.

    Article  PubMed  Google Scholar 

  21. Snyder W, Cook M, Nasset E, Karhansen L, Howells G, Tipton I Report of the task group on reference men. Oxford, United Kingdom Pergamon Press. 1975;23.

  22. Shankaran M, Czerwieniec G, Fessler C, Wong PA, Killion S, Turner SM, et al. Dilution of oral D3 -Creatine to measure creatine pool size and estimate skeletal muscle mass: development of a correction algorithm. J Cachexia Sarcopenia Muscle. 2018;9:540–6.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Heymsfield SB, Ebbeling CB, Zheng J, Pietrobelli A, Strauss BJ, Silva AM, et al. Multi-component molecular-level body composition reference methods: Evolving concepts and future directions. Obes Rev. 2015;16:282–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kondo E, Shiose K, Yamada Y, Osawa T, Sagayama H, Motonaga K, et al. Effect of thoracic gas volume changes on body composition assessed by air displacement plethysmography after rapid weight loss and regain in elite collegiate wrestlers. Sport. 2019;7.

  25. Sagayama H, Kondo E, Tanabe Y, Ohnishi T, Yamada Y, Takahashi H. Bone mineral density in male weight-classified athletes is higher than that in male endurance-athletes and non-athletes. Clin Nutr ESPEN. 2020;36:106–10.

    Article  PubMed  Google Scholar 

  26. Sagayama H, Yamada Y, Ichikawa M, Kondo E, Yasukata J, Tanabe Y, et al. Evaluation of fat-free mass hydration in athletes and non-athletes. Eur J Appl Physiol. 2020;120:1179–88.

    Article  CAS  PubMed  Google Scholar 

  27. Sagayama H, Yoshimura E, Yamada Y, Ichikawa M, Ebine N, Higaki Y, et al. Effects of rapid weight loss and regain on body composition and energy expenditure. Appl Physiol Nutr Metab. 2014;39:21–7.

    Article  CAS  PubMed  Google Scholar 

  28. Sagayama H, Shizuma K, Toguchi M, Mizuhara H, Machida Y, Yamada Y, et al. Effect of the Health Tourism weight loss programme on body composition and health outcomes in healthy and excess-weight adults. Br J Nutr. 2018;119:1133–1141.

    Article  CAS  PubMed  Google Scholar 

  29. Sagayama H, Racine NM, Shriver TC, Schoeller DA. Comparison of isotope ratio mass spectrometry and cavity ring-down spectroscopy procedures and precision of the doubly labeled water method in different physiological specimens. Rapid Commun Mass Spectrom. 2021;35:1–9.

    Article  Google Scholar 

  30. Hill DK. The location of creatine phosphate in frog’s striated muscle. J Physiol. 1962;164:31–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Edström L, Hultman E, Sahlin K, Sjöholm H. The contents of high-energy phosphates in different fibre types in skeletal muscles from rat, guinea-pig and man. J Physiol. 1982;332:47–58.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kondo E, Sagayama H, Yamada Y, Shiose K, Osawa T, Motonaga K, et al. Energy deficit required for rapid weight loss in elite collegiate wrestlers. Nutrients. 2018;10.

  33. Miyauchi S, Oshima S, Asaka M, Kawano H, Torii S, Higuchi M. Organ size increases with weight gain in power-trained athletes. Int J Sport Nutr Exerc Metab. 2013;23:617–23.

    Article  PubMed  Google Scholar 

  34. Müller MJ, Bosy-Westphal A, Braun W, Wong MC, Shepherd JA, Heymsfield SB. What Is a 2021 reference body? Nutrients 2022;14:1–18.

    Article  Google Scholar 

  35. Duchowny KA, Peters KE, Cummings SR, Orwoll ES, Hoffman AR, Ensrud KE, et al. Association of change in muscle mass assessed by D3-creatine dilution with changes in grip strength and walking speed. J Cachexia Sarcopenia Muscle. 2020;11:55–61.

    Article  PubMed  Google Scholar 

  36. Watt KKO, Garnham AP, Snow RJ. Skeletal muscle total creatine content and creatine transporter gene expression in vegetarians prior to and following creatine supplementation. Int J Sport Nutr Exerc Metab. 2004;14:517–31.

    Article  CAS  PubMed  Google Scholar 

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We wish to thank the volunteers who participated in this study and their coaches. The authors also thank Mr. Tsuyoshi Sakamoto for his advice on the MRI analysis.


This investigation was mainly supported by the Research Fellowships from the JSPS KAKENHI (16J11877 and 20K19563 to HS, 19H04017 to HT, and 18H03164 to YY). This data analysis and publication were supported by a grant 2022(I)1 from the Advanced Research Initiative for Human High Performance (ARIHHP), University of Tsukuba.

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Authors and Affiliations



HS, YY, and HT planned the study. HS, EK, TY, TO, and HT collected data. MS, EN, WE, MH, JY, and HY performed specimen and data analyses. HS, AU, and YY conducted statistical analyses. HS prepared illustrations. HS, EK, AU, and YY drafted the manuscript. All authors interpreted the results and revised and approved the final version of the manuscript.

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Correspondence to Hiroyuki Sagayama.

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Competing interests

WJE and MH are listed as co-inventors on patents for the D3-creatine dilution method. However, they do not control the IP nor do they derive any income from the use of this method. The other authors declare no competing interests.

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Sagayama, H., Yamada, Y., Kondo, E. et al. Skeletal muscle mass can be estimated by creatine (methyl‐d3) dilution and is correlated with fat-free mass in active young males. Eur J Clin Nutr 77, 393–399 (2023).

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