Does Human Skeletal Muscle Possess an Epigenetic Memory of Testosterone?

This project's primary aim of this double-blinded, randomised, placebo-controlled trial is to investigate whether short-term testosterone administration +/- resistance exercise training induces a muscle memory response that can lead to longer-lasting benefits in aged human skeletal muscle.

The investigators will provide older men with the anabolic hormone, testosterone or placebo, with or without resistance training, followed by a period of testosterone abstinence and detraining, followed by a subsequent repeated period of resistance training (retraining). This will help determine if earlier encounters with short-term testosterone administration can be "remembered" and if adaptation to later retraining can be enhanced as a consequence of encountering testosterone earlier.

Study Overview

• Status: Completed
• Conditions: Healthy Aging; Testosterone Deficiency; Age-Related Sarcopenia
• Intervention / Treatment: Drug: Saline; Drug: Testosterone Undecanoate; Drug: Saline + Resistance exercise training; Drug: Testosterone Undecanoate + Resistance exercise training

• Study Type: Interventional
• Enrollment (Actual): 45
• Phase: Phase 2; Phase 3

Contacts and Locations

Study Locations

• Norway
  • Oslo County
    • Oslo, Oslo County, Norway, 0863
      • Norwegian School of Sport Sciences

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Sedentary males
• 55-70 years old
• Serum testosterone levels >8 nmol/L measured in the morning
• Without any known illness, disease or other conditions
• Undergone screening through medical questionnaire, physical examination, routine blood tests and urine sample
• Written informed consent received

Exclusion Criteria:

• Current or previous participation in a formal exercise regime
• A BMI < 18 or > 30 kg·m2
• Hypersensitivity to the study drug or to any of its constituents
• Active cardiovascular disease: uncontrolled hypertension (BP > 160/100 mmHg), angina, heart failure (class III/IV), arrhythmia, right to left cardiac shunt, recent cardiac event
• Family history of early (<55y) death from cardiovascular disease
• Haematocrit >50%
• Malignancy
• Prostate-specific antigen (PSA) >4 ng/mL
• Lower urinary tract symptoms
• Taking beta-adrenergic blocking agents, statins, non-steroidal anti-inflammatory drugs
• Cerebrovascular disease: previous stroke, aneurysm (large vessel or intracranial), epilepsy
• Respiratory diseases including: pulmonary hypertension, chronic obstructive pulmanary disease (COPD), asthma, sleep apnoea
• Metabolic disease: hyper and hypo parathyroidism, untreated hyper and hypothyroidism, Cushing's disease, type 1 or 2 diabetes
• Active inflammatory bowel or renal disease
• Current or previous steroid treatment or hormone replacement therapy
• Clotting dysfunction
• Musculoskeletal or neurological disorders
• Alcohol or drug abuse
• Receiving oral anticoagulants
• Serum testosterone levels above the reference range for 50 year olds (>32 nmol/L) (Bjerner et al., 2009) measured in the morning 1

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Placebo Comparator: Placebo; The placebo group will complete 10-week treatment period where they continue with their regular habitual daily physical activity and receive two placebo (saline) injections (at baseline and week 3). They will then undergo a 12-week period with no treatment and no training, where they just do their regular habitual daily physical activity. Before they undertake a period of structured, progressive resistance training for 10-weeks. Questionnaires, physiological and psychological measures, skeletal muscle biopsies and blood samples will be taken at time points of: Baseline (week 0); Treatment period (week 10); Detraining and placebo abstinence (week 22); Retraining (week 32)	Drug: Saline; Two placebo injections one at baseline and one week 3.; Other Names: Placebo
Experimental: Testosterone Undecanoate; The testosterone group will complete 10-week treatment period where they continue with their regular habitual daily physical activity and receive two testosterone undecanoate (Nebido) injections (1000 mg/4 ml at baseline and 500 mg/2 ml week 3). They will then undergo a 12-week period with no treatment and no training, where they just do their regular habitual daily physical activity. Before they undertake a period of structured, progressive resistance training for 10-weeks. Questionnaires, physiological and psychological measures, skeletal muscle biopsies and blood samples will be taken at time points of: Baseline (week 0); Treatment period (week 10); Detraining and testosterone abstinence (week 22); Retraining (week 32)	Drug: Testosterone Undecanoate; Two testosterone undecanoate injections, 1000 mg/4 ml at baseline, 500 mg/2 ml at week 3.; Other Names: Nebido, Grünenthal (Grünenthal Norway AS)
Placebo Comparator: Resistance exercise training + Placebo; The resistance exercise training + placebo group will complete 10-week treatment period where they undergo a period of structured, progressive resistance training and receive two placebo (saline) injections (at baseline and week 3). They will then undergo a 12-week period with no treatment and no training, where they return to their regular habitual daily physical activity. Before they undertake a second period of structured, progressive resistance training for 10-weeks. Questionnaires, physiological and psychological measures, skeletal muscle biopsies and blood samples will be taken at time points of: Baseline (week 0); Treatment period (week 10); Detraining and placebo abstinence (week 22); Retraining (week 32)	Drug: Saline + Resistance exercise training; Two placebo injections one at baseline and one week 3 and10 weeks of supervised, structured, progressive resistance training.; Other Names: Placebo + Resistance exercise training
Experimental: Resistance exercise training + Testosterone Undecanoate; The resistance exercise training + testosterone group will complete 10-week treatment period where they undergo a period of structured, progressive resistance training and receive two testosterone undecanoate (Nebido) injections (1000 mg/4 ml at baseline and 500 mg/2 ml week 3). They will then undergo a 12-week period with no treatment and no training, where they return to their regular habitual daily physical activity. Before they undertake a second period of structured, progressive resistance training for 10-weeks. Questionnaires, physiological and psychological measures, skeletal muscle biopsies and blood samples will be taken at time points of: Baseline (week 0); Treatment period (week 10); Detraining and testosterone abstinence (week 22); Retraining (week 32)	Drug: Testosterone Undecanoate + Resistance exercise training; Two testosterone undecanoate injections, 1000 mg/4 ml at baseline, 500 mg/2 ml at week 3 and 10 weeks of supervised, structured, progressive resistance training.; Other Names: Nebido, Grünenthal (Grünenthal Norway AS) + Resistance exercise training

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Fat-free mass	Change and differences in fat-free mass (g) measured by dual x-ray absorptiometry (DEXA).	Baseline and weeks 10, 22, 32
Skeletal muscle size and cross-sectional area (CSA)	Change and differences in skeletal muscle size and CSA measured by ultrasound	Baseline and week 5,10, 16, 22, 27, 32
Skeletal muscle fibre CSA	Change and differences in skeletal muscle fibre CSA measure determined by immunohistochemistry	Baseline and weeks 10, 22, 32

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
DNA methylation in skeletal muscle and blood	Methylation measured in difference/fold change values relative to appropriate controls.	Baseline and weeks 10, 22, 32
Gene expression in skeletal muscle and blood	Gene expression measured in difference/fold change values relative to appropriate controls.	Baseline and weeks 10, 22, 32
Myonuclei	Change and differences in number of myonuclei determined by immunohistochemistry	Baseline and weeks 10, 22, 32
Satellite cells	Change and differences in number of satellite cells determined by immunohistochemistry	Baseline and weeks 10, 22, 32
Isometric muscle strength	Change and differences in peak muscle strength (N) using isokinetic dynamometry	Baseline and weeks 5, 10, 16, 22, 27, 32
Dynamic muscle strength	Change and differences in 1-repetition maximum	Baseline and weeks 5, 10, 16, 22, 27, 32
Muscle force-velocity profiling	Change and differences in force (N) and velocity (m/s) derived from a 10-repetition FV-test	Baseline and weeks 5, 10, 16, 22, 27, 32

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Skeletal muscle stiffness	Change and differences in skeletal muscle stiffness measured by shear wave ultrasonography	Baseline and weeks 10, 22, 32
Skeletal muscle tissue characteristics	Change and differences in skeletal muscle tissue characteristics determined by immunohistochemistry of muscle biopsies	Baseline and weeks 10, 22, 32
Bone mineral density	Change and differences in bone mineral density (g/cm2) measured by DEXA	Baseline and weeks 10, 22, 32
Bone health	Change and differences in bone health determined by bone health biomarkers in blood	Baseline and weeks 10, 22, 32
Blood parameters	Change and differences in steroid hormones in blood (testosterone, androstenediol, estradiol, and other relevant steroid markers, reproductive hormones (LH, FSH), binding protein (SHBG), cholesterol (total cholesterol, LDL, HDL), and PSA level, and endocrine biomarkers (IGF-1 and P-III-NP).	Baseline and weeks 10, 22, 32
Aging males symptoms	Change and differences in Aging male symptoms score (1="none", 5="extremely severe")	Baseline and weeks 5, 10, 16, 22, 27, 32
Well-being	Change and differences in WHO5 well-being index score (0="at not time", 5="all of the time")	Baseline and weeks 5, 10, 16, 22, 27, 32
Psychological distress	Change and differences in SCL-10 symptoms score (1="not at all", 4="extremely")	Baseline and weeks 5, 10, 16, 22, 27, 32
Fatigue	Change and differences in shortened fatigue questionnaire score (1="yes, that is true", 7="no, that is not true")	Baseline and weeks 5, 10, 16, 22, 27, 32
Sleep	Change and differences in Jenkins sleep scale score (0="not at all", 5="22-31days")	Baseline and weeks 5, 10, 16, 22, 27, 32
Sexual function	Change and differences in sexual function score (items from Health-related quality of life (HRQOL) questionnaire), (	Baseline and weeks 5, 10, 16, 22, 27, 32
Body perception	Change and differences in Body Perception Questionnaire very short form score (1=never, 5=always)	Baseline and weeks 5, 10, 16, 22, 27, 32
Anger	Change and differences in The State Anger subscale of STAXI-2score (0="not at all", 3="very much")	Baseline and weeks 5, 10, 16, 22, 27, 32
Mania	Change and differences in Altman Self-Rating Mania Scale (ASRM) score	Baseline and weeks 5, 10, 16, 22, 27, 32
Suicide thoughts	Change and differences in Suicide thoughts from Montgomery and Åsberg Depression Rating Scale	Baseline and weeks 5, 10, 16, 22, 27, 32
Exercise effort	Change and differences in rating of perceived exertion for effort (Borg CR-10 RPE)	Baseline and weekly up to week 10, and weekly from week 22 up to week 32
Exercise discomfort	Change and differences in rating of perceived exertion for discomfort (sRPD) score	Baseline and weekly up to week 10, and weekly from week 22 up to week 32
Session pleasure and displeasure	Change and differences in perceived pleasure/displeasure with the training session using the pleasure/displeasure feeling scale (sPDF), (-5=very bad", 5="very good")	Baseline and weekly up to week 10, and weekly from week 22 up to week 32
Exercise enjoyment	Change and differences in exercise enjoyment scale score (1="not at all", 7=extraordinary")	Baseline and weekly up to week 10, and weekly from week 22 up to week 32
Blood volume, haemoglobin and lean body mass	To investigate the association between blood volume, haemoglobin mass and lean body mass measured by carbon monoxide (CO) rebreathing and DEXA.	Baseline and weeks 10, 22, 32
Cognitive function	Change and differences in cognitive function measured by emotion recognition tasks and cognitive reflection tasks	Baseline and weeks 5, 10, 16, 22, 27, 32
Exercise motivation	Change and differences in perceived motivation score and motivation type	Baseline and weekly up to week 10, and weekly from week 22 up to week 32
Reps in reserve	Change and differences in the ability to predict reps in reserve	Baseline and weekly up to week 10, and weekly from week 22 up to week 32

Collaborators and Investigators

• Sponsor: Norwegian School of Sport Sciences
• Collaborators: Oslo University Hospital

Publications and helpful links

General Publications

• Egner IM, Bruusgaard JC, Eftestol E, Gundersen K. A cellular memory mechanism aids overload hypertrophy in muscle long after an episodic exposure to anabolic steroids. J Physiol. 2013 Dec 15;591(24):6221-30. doi: 10.1113/jphysiol.2013.264457. Epub 2013 Oct 28.
• Seaborne RA, Strauss J, Cocks M, Shepherd S, O'Brien TD, van Someren KA, Bell PG, Murgatroyd C, Morton JP, Stewart CE, Sharples AP. Human Skeletal Muscle Possesses an Epigenetic Memory of Hypertrophy. Sci Rep. 2018 Jan 30;8(1):1898. doi: 10.1038/s41598-018-20287-3.
• Turner DC, Seaborne RA, Sharples AP. Comparative Transcriptome and Methylome Analysis in Human Skeletal Muscle Anabolism, Hypertrophy and Epigenetic Memory. Sci Rep. 2019 Mar 12;9(1):4251. doi: 10.1038/s41598-019-40787-0.
• Sharples AP, Turner DC. Skeletal muscle memory. Am J Physiol Cell Physiol. 2023 Jun 1;324(6):C1274-C1294. doi: 10.1152/ajpcell.00099.2023. Epub 2023 May 8.
• Gharahdaghi N, Rudrappa S, Brook MS, Idris I, Crossland H, Hamrock C, Abdul Aziz MH, Kadi F, Tarum J, Greenhaff PL, Constantin-Teodosiu D, Cegielski J, Phillips BE, Wilkinson DJ, Szewczyk NJ, Smith K, Atherton PJ. Testosterone therapy induces molecular programming augmenting physiological adaptations to resistance exercise in older men. J Cachexia Sarcopenia Muscle. 2019 Dec;10(6):1276-1294. doi: 10.1002/jcsm.12472. Epub 2019 Sep 30.
• Sharples AP, Polydorou I, Hughes DC, Owens DJ, Hughes TM, Stewart CE. Skeletal muscle cells possess a 'memory' of acute early life TNF-alpha exposure: role of epigenetic adaptation. Biogerontology. 2016 Jun;17(3):603-17. doi: 10.1007/s10522-015-9604-x. Epub 2015 Sep 8.
• Wen Y, Dungan CM, Mobley CB, Valentino T, von Walden F, Murach KA. Nucleus Type-Specific DNA Methylomics Reveals Epigenetic "Memory" of Prior Adaptation in Skeletal Muscle. Function (Oxf). 2021 Aug 5;2(5):zqab038. doi: 10.1093/function/zqab038. eCollection 2021.

Study record dates

Study Major Dates

• Study Start (Actual): September 1, 2024
• Primary Completion (Actual): December 22, 2025
• Study Completion (Actual): February 6, 2026

Study Registration Dates

• First Submitted: June 16, 2023
• First Submitted That Met QC Criteria: July 19, 2023
• First Posted (Actual): July 28, 2023

Study Record Updates

• Last Update Posted (Actual): March 6, 2026
• Last Update Submitted That Met QC Criteria: March 5, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: Healthy aging; Anti-Doping; Preventing Age-related Sarcopenia; Overcoming Anabolic Resistance
• Additional Relevant MeSH Terms: Nervous System Diseases; Neurologic Manifestations; Neuromuscular Manifestations; Pathological Conditions, Anatomical; Muscular Atrophy; Atrophy; Sarcopenia; Inorganic Chemicals; Chlorine Compounds; Sodium Compounds; Chlorides; Hydrochloric Acid; Sodium Chloride; testosterone undecanoate
• Other Study ID Numbers: TESTO-MEM

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: No identifiable information will be included in any publication. Genome-Wide DNA Methylation and Gene Expression will be deposited with full open access on Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) an internationally recognised database. GEO data derived from human samples will be anonymous, with no identifiable information. GEO data will be deposited at the same time as a publication. All non-identifiable results from the project will be ultimately published in peer-reviewed journals. In addition, all image files, raw excel/ .csv / txt /word files for any of the other analyses described above in the methods will be available as either supplementary files on the publisher's website or fully accessible on request to the corresponding author/authors.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No