Women’s Health Bulletin

Women’s Health Bulletin

Effects of Eight Weeks of Functional Training with Blood Flow Restriction and Betaine Supplementation on Follistatin, Myostatin, and Functional Outcomes in Older Women: A Randomized Controlled Trial Protocol

Document Type : Study Protocol

Authors
Faezeh Aliakbari Jam 1
Elham Shakoor 1
Farzaneh Zeynali 1
Ghorbangol Ashabi 2
Samaneh Seyedi 2
Katsuhiko Suzuki 3
1 Department of Exercise Physiology, Faculty of Sport Sciences, Alzahra University, Tehran, Iran
2 Department of Physiology, School of Medicine, Tehran University of Medical Science, Tehran, Iran
3 Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa 359-1192, Japan
10.30476/whb.2026.109558.1383
Abstract
Background: Sarcopenia is characterized by age-related declines in muscle mass and function. Functional training with blood flow restriction (BFR) and betaine supplementation may improve anabolic adaptations; however, their combined effects in elderly women remain unclear. Therefore, this study aims to investigate their effects on serum follistatin and myostatin levels in elderly women.
Methods: This randomized, double-blind, placebo-controlled clinical trial protocol will enroll 56 community-dwelling older women (≥60 years) in Tehran, Iran. Participants will be randomized (1:1:1:1 allocation via a computer-generated sequence with allocation concealment) into four parallel groups (n=14 each) including functional training with blood flow restriction plus betaine (FBFR+B), functional training with blood flow restriction plus placebo (FBFR+P), betaine supplementation alone (B; 4 g/day), or non-exercising control (CON) for 8 weeks (exercise 3 sessions/week). Primary outcomes (plasma follistatin and myostatin measured by enzyme-linked immunosorbent assay) and secondary outcomes [muscle strength via 1RM bench/ leg press, functional performance via Timed Up-and-Go (TUG) and 10-Meter Walk Test (10-mWT), health-related quality of life (WHOQOL-BREF), and pain characteristics (McGill Pain Questionnaire)] will be evaluated at baseline and 48 hours following the 8-week intervention. Between-group differences will be analyzed using ANCOVA adjusted for baseline values, with significance set at P<0.05.
Discussion: This study will evaluate the effects of functional training combined with blood flow restriction (BFR) and betaine supplementation on serum follistatin and myostatin levels in elderly women. Findings from this research may inform future guidelines for non-pharmacological interventions to preserve muscle health, preserve independence, and manage sarcopenia in aging populations.
Keywords
Sarcopenia
Betaine
Follistatin
Myostatin
Resistance Training
1. Behr LC, Simm A, Kluttig A, Großkopf AG. 60 years of healthy aging: on definitions, biomarkers, scores and challenges. Ageing Res Rev. 2023;88:101934. doi: 10.1016/j.arr.2023.101934. PubMed PMID: 37059401. ##
2. Falchetta G, De Cian E, Sue Wing I, Carr D. Global projections of heat exposure of older adults. Nat Commun. 2024;15(1):3678. doi: 10.1038/s41467-024-47197-5. PubMed PMID: 38744815; PubMed Central PMCID: PMC11094092. ##
3. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: An expanding universe. Cell. 2023;186(2):243-278. doi: 10.1016/j.cell.2022.11.001. PubMed PMID: 36599349. ##
4. Kirk B, Cawthon PM, Arai H, Ávila-Funes JA, Barazzoni R, Bhasin S, et al. The Conceptual Definition of Sarcopenia: Delphi Consensus from the Global Leadership Initiative in Sarcopenia (GLIS). Age Ageing. 2024;53(3):afae052. doi: 10.1093/ageing/afae052. PubMed PMID: 38520141; PubMed Central  PMCID: PMC10960072. ##
5. Lan XQ, Deng CJ, Wang QQ, Zhao LM, Jiao BW, Xiang Y. The role of TGF-β signaling in muscle atrophy, sarcopenia and cancer cachexia. General and Comparative Endocrinology. 2024;353:114513. doi: 10.1016/j.ygcen.2024.114513. ##
6. Samali SA, Hosseini SF, Mohammadi Y, Sadri F, Rezaei Z. Myostatin inhibitors in sarcopenia treatment: A comprehensive review of mechanisms, efficacy and future directions. Mol Biol Rep. 2026;53(1):224. doi: 10.1007/s11033-025-11390-6. PubMed PMID: 41460393. ##
7. Wetzlich B, Nyakundi BB, Yang J. Therapeutic applications and challenges in myostatin inhibition for enhanced skeletal muscle mass and functions. Mol Cell Biochem. 2025;480(3):1535-1553. doi: 10.1007/s11010-024-05120-y. PubMed PMID: 39340593; PubMed Central PMCID: PMC11842502. ##
8. Fabero-Garrido R, Gragera-Vela M, Del Corral T, Hernández-Martín M, Plaza-Manzano G, López-de-Uralde-Villanueva I. Effects of Low-Load Blood Flow Restriction Training on Muscle Anabolism Biomarkers and Thrombotic Biomarkers Compared with Traditional Training in Healthy Adults Older Than 60 Years: Systematic Review and Meta-Analysis. Life. 2024;14(3):411. doi: 10.3390/life14030411. PubMed PMID: 38541735; PubMed Central PMCID: PMC10971244. ##
9. Tan Z, Jiang Y, Candow DG, Castagna C, Wang X, Zheng H. Optimizing prescription of resistance training for body composition, muscle strength, and physical performance in older adults with sarcopenia: a systematic review and meta-analysis. Eur Rev Aging Phys Act. 2026;23(1):8. doi: 10.1186/s11556-025-00399-2. PubMed PMID: 41566419; PubMed Central PMCID: PMC12908291. ##
10. American College of Sports Medicine, Chodzko-Zajko WJ, Proctor DN, Fiatarone Singh MA, Minson CT, Nigg CR, Salem GJ, Skinner JS. American College of Sports Medicine position stand. Exercise and physical activity for older adults. Med Sci Sports Exerc. 2009;41(7):1510-30. doi: 10.1249/MSS.0b013e3181a0c95c. PubMed PMID: 19516148. ##
11. Papa EV, Dong X, Hassan M. Resistance training for activity limitations in older adults with skeletal muscle function deficits: a systematic review. Clin Interv Aging. 2017;12:955-961. doi: 10.2147/CIA.S104674. PubMed PMID: 28670114; PubMed Central PMCID: PMC5479297. ##
12. Labata-Lezaun N, Llurda-Almuzara L, González-Rueda V, López-de-Celis C, Cedeño-Bermúdez S, Bañuelos-Pago J, et al. Effectiveness of Blood Flow Restriction Training on Muscle Strength and Physical Performance in Older Adults: A Systematic Review and Meta-analysis. Arch Phys Med Rehabil. 2022;103(9):1848-1857. doi: 10.1016/j.apmr.2021.12.015. PubMed PMID: 35026149. ##
13. Ramadhan NA, Tinduh D, Nugraheni N, Narasinta I, Subadi I, Setiawan A, et al. Low-Intensity Resistance Exercise with Blood Flow Restriction Improves Balance in Elderly Women: A Comparative Study on the Lower Quarter Y-Balance Test. Salud, Ciencia y Tecnologia. 2025;5:1148. doi: 10.56294/saludcyt20251148. ##
14. Espinosa-Méndez CM, Flores-Moreno PJ, Reyes-Espejel I, Ortega-González JA. Strength Training with Blood Flow Restriction: Effect on Factors Associated with Sarcopenia in Older Women. Sport Mont. 2025;23(2):43-8. doi: 10.26773/smj.250607. ##
15. Pazokian F, Amani-Shalamzari S, Rajabi H. Effects of functional training with blood occlusion on the irisin, follistatin, and myostatin myokines in elderly men. Eur Rev Aging Phys Act. 2022;19(1):27. doi: 10.1186/s11556-022-00308-x. PubMed PMID: 36324082. ##
16. Zawieja E, Machek S, Zanchi NE, Cholewa J, Woźniewicz M. Effects of chronic betaine supplementation on exercise performance: Systematic review and meta-analysis. J Sports Sci. 2024;42(22):2131-2144. doi: 10.1080/02640414.2024.2423578. PubMed PMID: 39514262. ##
17. Van Every DW, Plotkin DL, Delcastillo K, Cholewa J, Schoenfeld BJ. Betaine supplementation: A critical review of its efficacy for improving muscle strength, power, and body composition. Strength & Conditioning Journal. 2021;43(4):53-61. doi: 10.1519/SSC.0000000000000622. ##
18. Machek SB, Harris DR, Zawieja EE, Heileson JL, Wilburn DT, Radziejewska A, et al. The Impacts of Combined Blood Flow Restriction Training and Betaine Supplementation on One-Leg Press Muscular Endurance, Exercise-Associated Lactate Concentrations, Serum Metabolic Biomarkers, and Hypoxia-Inducible Factor-1α Gene Expression. Nutrients. 2022;14(23):5040. doi: 10.3390/nu14235040. PubMed PMID: 36501070; PubMed Central PMCID: PMC9739923. ##
19. Lim ZX, Goh J. Effects of blood flow restriction (BFR) with resistance exercise on musculoskeletal health in older adults: a narrative review. Eur Rev Aging Phys Act. 2022;19(1):15. doi: 10.1186/s11556-022-00294-0. PubMed PMID: 35725379; PubMed Central PMCID: PMC9208167. ##
20. Chen S, He T, Chen J, Wen D, Wang C, Huang W, et al. Betaine delays age‐related muscle loss by mitigating Mss51‐induced impairment in mitochondrial respiration via Yin Yang1. J Cachexia Sarcopenia Muscle. 2024;15(5):2104-2117. doi: 10.1002/jcsm.13558. PubMed PMID: 39187977; PubMed Central PMCID: PMC11446699. ##
21. Chan AW, Tetzlaff JM, Altman DG, Laupacis A, Gøtzsche PC, Krleža-Jerić K, et al. SPIRIT 2013 statement: defining standard protocol items for clinical trials. Ann Intern Med. 2013;158(3):200-7. doi: 10.7326/0003-4819-158-3-201302050-00583. PubMed PMID: 23295957; PubMed Central PMCID: PMC5114123. ##
22. Hoffmann TC, Glasziou PP, Boutron I, Milne R, Perera R, Moher D, et al. Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014;348:g1687. doi: 10.1136/bmj.g1687. PubMed PMID: 24609605. ##
23. Brzycki M. Strength Testing—Predicting a One-Rep Max from Reps-to-Fatigue. Journal of Physical Education, Recreation & Dance. 1993;64(1):88-90. doi: 10.1080/07303084.1993.10606684. ##
24. Loenneke JP, Allen KM, Mouser JG, Thiebaud RS, Kim D, Abe T, et al. Blood flow restriction in the upper and lower limbs is predicted by limb circumference and systolic blood pressure. Eur J Appl Physiol. 2015;115(2):397-405. doi: 10.1007/s00421-014-3030-7. PubMed PMID: 25338316. ##
25. Khanmohammadi F, Shakoor E, Ranjbar K, Ashabi G. Effects of eight weeks of functional training with and without blood flow restriction on serum irisin, IGF-1, and lipid profile in elderly women: A randomized controlled clinical trial. Journal of Exercise & Organ Cross Talk. 2026;6(3):156-166. doi: 10.22122/jeoct.2026.595101.1222. ##
26. Sadeghi H, Jehu DA, Daneshjoo A, Shakoor E, Razeghi M, Amani A, et al. Effects of 8 Weeks of Balance Training, Virtual Reality Training, and Combined Exercise on Lower Limb Muscle Strength, Balance, and Functional Mobility Among Older Men: A Randomized Controlled Trial. Sports Health. 2021;13(6):606-612. doi: 10.1177/1941738120986803. PubMed PMID: 33583253; PubMed Central PMCID: PMC8558995. ##
27. Thomson D, Liston M, Gupta A. Is the 10 metre walk test on sloped surfaces associated with age and physical activity in healthy adults? Eur Rev Aging Phys Act. 2019;16:11. doi: 10.1186/s11556-019-0219-0. PubMed PMID: 31360260; PubMed Central PMCID: PMC6639969. ##
28. Nedjat S, Montazeri A, Holakouie K, Mohammad K, Majdzadeh R. Psychometric properties of the Iranian interview-administered version of the World Health Organization's Quality of Life Questionnaire (WHOQOL-BREF): a population-based study. BMC Health Serv Res. 2008;8:61. doi: 10.1186/1472-6963-8-61. PubMed PMID: 18366715; PubMed Central PMCID: PMC2287168. ##
29. Adelmanesh F, Jalali A, Attarian H, Farahani B, Ketabchi SM, Arvantaj A, et al. Reliability, validity, and sensitivity measures of expanded and revised version of the short-form McGill Pain Questionnaire (SF-MPQ-2) in Iranian patients with neuropathic and non-neuropathic pain. Pain Med. 2012;13(12):1631-6. doi: 10.1111/j.1526-4637.2012.01517.x. PubMed PMID: 23137190. ##
30. Radaelli R, Rech A, Molinari T, Markarian AM, Petropoulou M, Granacher U, et al. Effects of Resistance Training Volume on Physical Function, Lean Body Mass and Lower-Body Muscle Hypertrophy and Strength in Older Adults: A Systematic Review and Network Meta-analysis of 151 Randomised Trials. Sports Med. 2025;55(1):167-192. doi: 10.1007/s40279-024-02123-z. PubMed PMID: 39405023. ##
31. Jochum D, Siegel SD, Plöschberger G, Fenske L, Warneke K. Resistance training effects on gait parameters in older adults—a systematic review with multilevel meta-analysis and meta-regression. Geroscience. 2026. doi: 10.1007/s11357-026-02156-4. PubMed PMID: 41811623. ##
32. Kechichian A, Lafrance S, Matifat E, Dube F, Lussier D, Benhaim P, et al. Multimodal Interventions Including Rehabilitation Exercise for Older Adults With Chronic Musculoskeletal Pain: A Systematic Review and Meta-analyses of Randomized Controlled Trials. J Geriatr Phys Ther. 2022;45(1):34-49. doi: 10.1519/JPT.0000000000000279. PubMed PMID: 34928239. ##
33.Knutzen KM, Pendergrast BA, Lindsey B, Brilla LR. The Effect of High Resistance Weight Training on Reported Pain in Older Adults. J Sports Sci Med. 2007;6(4):455-460. PubMed PMID: 24149478; PubMed Central PMCID: PMC3794485. ##