A comprehensive timeline: The development of Testosterone Replacement

Testosterone replacement therapy (TRT) isn’t a modern invention—it’s the product of nearly two centuries of scientific discovery, clinical trials, and pharmaceutical innovation. From Aristotle’s observations on castration to today’s subcutaneous microdosing protocols, the story of TRT reveals how medicine evolves: from crude empiricism through surgical experimentation, to precise chemistry, and finally to individualised, evidence-based practice.

Introduction

This timeline traces that journey for clinicians and informed patients alike. You’ll discover how ancient physicians unknowingly laid the groundwork for endocrinology, how 19th-century surgeons attempted (and failed) to rejuvenate ageing men through testicular transplants, and how three simultaneous breakthroughs in 1935 transformed testosterone from a philosophical concept into a reliable pharmaceutical. Most importantly, you’ll see how modern formulations—from gels and patches to subcutaneous microdosing and novel SARMs—have made TRT safer, more physiological, and increasingly tailored to individual patient needs.

Whether you’re a prescriber refining your approach, a patient understanding your treatment options, or a researcher exploring emerging therapies, this comprehensive timeline provides the clinical context and historical perspective needed to appreciate where TRT stands today—and where it’s heading.

Five key takeaways

1. Endocrinology emerged from observation, not invention: Berthold’s 1849 internal secretion hypothesis was the true inflection point. Before Berthold, transplantation experiments (Hunter, 1786) were curiosities without theoretical framework. Berthold’s explicit proposal that the testis released chemical substances into the bloodstream—rather than exerting effects via the nervous system—created the conceptual foundation for all modern hormone research. This shift from mechanism-agnostic observation to testable theory accelerated the entire field.

2. The transplantation era (1890–1935) was scientifically earnest but ultimately futile; chemistry rendered it obsolete overnight. Surgeons like Lydston documented meticulous physiological changes and drew reasonable conclusions about graft success and vascular proximity. Yet even their best work couldn’t compete with 1935’s triple breakthrough: Lacquer’s crystalline testosterone isolation and Butenandt/Ruzicka’s chemical synthesis. Once testosterone could be reliably produced in pure form, transplantation became unnecessary. This illustrates a broader principle: biological interventions often give way to pharmaceutical ones once the active principle is identified and synthesised.

3. Formulation innovation has been driven by bioavailability and compliance challenges, not by efficacy gaps. Native testosterone’s poor oral bioavailability prompted pellets (1930s), then 17-methyl testosterone (hepatotoxic), then esters (enanthate, cypionate, undecanoate), then transdermal patches and gels (1990s–2000s), and now subcutaneous microdosing and novel delivery systems (2020s). Each innovation solved a real clinical problem—hepatic first-pass metabolism, injection frequency, patient preference, or haematocrit elevation—rather than replacing an ineffective therapy. This pattern suggests future advances will continue targeting delivery, stability, and individualisation rather than the hormone itself.

4. Haematocrit elevation and polycythemia risk are formulation-dependent, not testosterone-dependent. The 2025 FDA clarification that approved TRT products don’t increase cardiovascular risk compared to placebo reflects maturation in pharmacovigilance. However, the recognition that intranasal testosterone produces significantly lower haematocrit elevation than intramuscular cypionate reveals a crucial nuance: the route and kinetics matter as much as the hormone. This supports the modern shift toward individualised formulation selection based on comorbidity profiles—a move from one-size-fits-all dosing to precision medicine.

5. Microdosing with subcutaneous testosterone cypionate represents a return to physiological principles, informed by decades of pharmacology. Pulsatile testosterone secretion in healthy men differs fundamentally from the pharmacokinetic peaks and troughs of traditional weekly or bi-weekly injections. Subcutaneous microdosing (10–50 mg, 1–3 times weekly) attempts to approximate this natural pattern whilst maintaining stable serum levels and reducing adverse effects. Although formal RCTs comparing microdosing to standard protocols remain limited, accumulating clinical experience and case series suggest real benefits in symptom stability and haematocrit management—a reminder that older physiological wisdom, combined with modern precision, often yields superior outcomes.

I. Ancient and classical origins: Early recognition of testicular function

The biological effects of the testes and testosterone have been recognised since ancient times [1]. Aristotle, one of antiquity’s greatest philosophers, was among the first to document the profound effects of castration on male development and behaviour. His early observations established a fundamental principle that would later form the basis of endocrinology: that the testes produced substances essential to male characteristics [1].

Throughout history, castration was practised extensively across diverse civilisations for various purposes. In Oriental imperial courts, particularly those of China and other Eastern kingdoms, eunuchs were deliberately created through castration and elevated to positions of political power and influence, serving as overseers in royal harems [1]. The practice extended into medieval and later European societies, where prepubertal boys were castrated to preserve their soprano voices for use in religious and operatic performances. These historical practices, while ethically troubling by modern standards, provided unintended evidence that testicular function was essential for maintaining male secondary sexual characteristics and physical vigour. The deliberate removal of testicular tissue across populations demonstrated the organ’s central role in human development and physiology, laying crucial groundwork for future scientific investigation [1].

II. The birth of endocrinology: From observation to experimentation (1786-1889)

The transition from mere observation to experimental science marked a pivotal moment in the history of testosterone research. In 1786, John Hunter, a renowned London surgeon and pioneer of experimental medicine, performed one of the first recorded testicular transplantation experiments [1]. Hunter transplanted testes from roosters (capons) into chickens, seeking to prove his hypothesis that an intangible “vital principle” governed life itself. While his philosophical framework later proved incorrect, his experiments inadvertently demonstrated the remarkable principle of germ cell transplantation and the potential for tissue survival across hosts. More importantly, Hunter’s work established the precedent for viewing the testes not merely as reproductive organs, but as sources of substances that could influence the entire organism [1].

The true father of modern endocrinology was Arnold Adolph Berthold, a German physiologist working in Göttingen in 1849. Berthold’s groundbreaking experiments involved castrating young roosters and then transplanting testicular tissue back into their abdominal cavities [1]. What distinguished Berthold’s work from Hunter’s was his explicit theoretical framework: he proposed that the testis operated through “internal secretion,” a revolutionary concept suggesting that organs could exert systemic effects by releasing chemical substances directly into the bloodstream rather than through nervous system pathways [1]. Berthold’s observations that transplanted testes maintained male characteristics in the castrated birds provided the first definitive evidence for what we now understand as endocrine function. His work remained relatively obscure for decades, but it established the fundamental principle upon which all subsequent hormone research would be built [2].

Charles-Edouard Brown-Séquard, a prominent Parisian physiologist, brought testicular extracts to public attention in 1889 through a series of self-experiments that captured both scientific and popular imagination [1].  Brown-Séquard, then 72 years old, injected himself with extracts derived from animal testicular tissue and reported remarkable improvements in vigour, strength, and sexual function [3]. While modern analysis suggests these effects were largely placebo in nature, his public demonstrations of testicular extract therapy marked the beginning of the modern era of hormone treatment and popularised the concept of rejuvenation through glandular substances  [1]. Brown-Séquard’s work, though scientifically limited, sparked enormous interest throughout Europe and America in the therapeutic potential of sex gland extracts and transplantation.

III. The era of testicular transplantation and organotherapy (1890-1935)

The late 19th and early 20th centuries witnessed an explosion of attempts to harness the rejuvenative power of the testes through transplantation and extract therapies. This period, often referred to as the age of organotherapy, represented both genuine scientific inquiry and considerable medical overreach. In 1915, G. Frank Lydston, a prominent Chicago urologist, conducted extensive research on testicular implantation and published a comprehensive 93-page monograph detailing his findings [4]. Lydston performed autotransplantation, heterotransplantation, and even cross-sex transplantation experiments, documenting physiological effects on everything from senility to chronic skin diseases [4].

Lydston’s work was notable not only for its scope but also for his detailed conclusions, which asserted that successful testicular implantation could temporarily or permanently treat various conditions, increase longevity, reduce arteriosclerosis, and boost physical and mental efficiency [4]. His 1916 publication in the Journal of the American Medical Association presented a case series supporting testicular implantation for the ageing male [5]. While Lydston’s enthusiasm sometimes exceeded the evidence, his systematic approach and documentation of physiological changes represented a significant advance in the field. His work influenced contemporary surgeons like Lespinasse, Morris, and Steinach, who pursued similar transplantation and rejuvenation techniques [3].

Sergio Voronoff, a Russian-French surgeon, became perhaps the most celebrated practitioner of testicular transplantation in the 1920s, performing over 5,000 procedures worldwide, often transplanting testicular material from animals (particularly primates) into human patients [1]. While Voronoff’s techniques initially captured public enthusiasm, their effectiveness was eventually disproven through rigorous clinical study [1].  Nevertheless, the Voronoff era demonstrated popular and medical interest in rejuvenation therapy and transplantation, even if the scientific basis proved inadequate. Other surgeons, including the infamous John R. Brinkley, who transplanted goat testicles into men, pushed transplantation into frank charlatanism, ultimately damaging the credibility of more serious researchers [3].

G. Frank Lydston’s work represented the height of transplantation-based therapy, documented through meticulous physiological observation, and his conclusions about the therapeutic potential of testicular implantation remained influential until biochemical isolation of testosterone itself rendered transplantation obsolete [3]. Lydston emphasised that properly selected cases could benefit from implantation, that graft success depended on proximity to vascular tissue, and that the testicular hormone acted as a stimulant, nutrient, and reconstructive agent [4]. His work bridged the gap between clinical observation and the emerging biochemical understanding of hormones.

IV. The chemical revolution: Isolation and synthesis of testosterone (1930s-1940s)

The true transformation of testosterone therapy came through chemistry rather than surgery. In 1935, three major discoveries occurred simultaneously that would revolutionise the field. Ernest Lacquer, working in Amsterdam, successfully isolated pure testosterone from bull testes in crystalline form [1]. In the same year, Adolf Butenandt in Göttingen and Leopold Ruzicka in Basel independently achieved the complete chemical synthesis of testosterone [1]. This convergence of biological isolation and chemical synthesis established testosterone as a well-characterised hormone that could be produced reliably and modified for therapeutic use.

The early phases of modern androgen therapy began in 1935 following these discoveries, marking the transition from biological to pharmaceutical approaches [1]. However, researchers quickly discovered that native testosterone was poorly bioavailable when administered orally due to rapid hepatic metabolism. This challenge spurred the development of alternative formulations and delivery systems. Testosterone was initially compressed into subcutaneous pellets for sustained release, a method that provided relatively stable hormone levels but required surgical implantation [1]. Concurrently, researchers developed 17-methyl testosterone, an orally active derivative that could survive hepatic metabolism, though this compound later fell out of favour due to documented hepatotoxicity [1].

The recognition of specific hypogonadal syndromes during this period provided a clinical context for testosterone replacement. In the 1940s and 1950s, several landmark descriptions of male hypogonadism emerged: Klinefelter syndrome was described, followed by Kallmann syndrome, and then the De Castillo and Pasqualini syndrome [1]. These clinical descriptions of specific testicular and gonadotropin deficiency states provided precise patient populations for whom testosterone replacement therapy had clear indications, moving treatment from the realm of rejuvenation and anti-ageing to legitimate endocrinological therapy.

V. Development of modern formulations and delivery systems (1950s-1990s)

The 1950s marked a major advancement in testosterone therapy with the introduction of longer-acting injectable esters. Testosterone enanthate, an esterified form with extended duration of action, became the preferred therapeutic modality and remains widely used today [1]. The hydrophobic nature of these esters, determined by the length of their side chains, enabled depot formation in muscle tissue with sustained release into circulation. Testosterone propionate, with a shorter chain, produced shorter duration effects, while testosterone enanthate provided therapeutic coverage for several weeks [1]. The development of testosterone cypionate followed a similar pharmacokinetic profile to enanthate, offering clinicians flexibility in dosing intervals [6].

The 1950s and 1960s witnessed intense research focused on chemical modification of androgens to enhance their anabolic properties while minimising androgenic effects—a goal that led to the development of numerous synthetic anabolic steroids  [1]. While these compounds found limited clinical application in legitimate medicine, they fundamentally expanded the pharmacological toolkit available for testosterone replacement and anabolic therapy. Researchers discovered that altering the steroid nucleus or adding specific side chains could bias compounds toward anabolic versus androgenic effects, principles that persist in modern steroid development  [1].

The 1970s introduced testosterone undecanoate, an orally active ester that could be absorbed via the lymphatic system rather than undergo first-pass hepatic metabolism [1]. This represented a major advance for patients who required oral therapy or desired to avoid injections. The availability of testosterone undecanoate expanded options for patients with different preferences and medical circumstances, though its pharmacokinetics required careful management to maintain therapeutic levels [7]. By the 1980s and 1990s, researchers began developing transdermal delivery systems, first as scrotal patches and later as non-genital patches and gels, providing patients with continuous, physiological testosterone exposure that more closely mimicked the diurnal variation of natural testosterone production [6].

VI. Contemporary era: Transdermal systems, novel formulations, and microdosing (1990s-Present)

The introduction of transdermal testosterone delivery revolutionised the clinical practice of testosterone replacement therapy. In the 1990s and 2000s, FDA-approved transdermal patches and gels became available, offering non-invasive alternatives to injections and oral therapy [8]. The first-generation Testoderm scrotal patches delivered testosterone through the highly permeable scrotal skin, though the requirement to shave the area and risk of skin irritation limited patient acceptance [6]. Non-genital transdermal patches like Androderm provided improved ease of use, and later testosterone gels such as Testim and AndroGel offered convenient daily application to the shoulders, arms, and abdomen with pharmacokinetic profiles that produced relatively stable mid-range testosterone levels [6].

The transdermal gel formulations fundamentally changed patient care and compliance with testosterone therapy. Clinical trials demonstrated that 12-month treatment with transdermal gels maintained adequate testosterone levels while producing statistically significant improvements in lean muscle mass, sexual motivation, and spontaneous erections, with minimal application-site reactions compared to earlier patch formulations [6]. The development of more physiological delivery systems addressed the major limitation of injectable esters, which created significant fluctuations in testosterone levels over the treatment cycle, sometimes causing mood swings and inconsistent symptom response  [1].

Recent decades have seen continued innovation in the design of testosterone formulations. Oral testosterone undecanoate formulations (Andriol, Jatenzo, Tlando, Kyzatrex) utilising novel delivery technologies, such as self-emulsifying drug delivery systems, have improved bioavailability and therapeutic reliability compared to earlier oral agents [7]. Long-acting injectable preparations using sophisticated lipid matrix technology provide testosterone coverage extending to 10-11 weeks between injections [6]. In 2025, the FDA approved Azmiro, a single-dose injectable formulation of testosterone cypionate in prefilled syringes and vials, representing the latest innovation in injectable therapy [9].

Subcutaneous injection of testosterone esters has emerged as a practical alternative to intramuscular injection, offering easier self-administration with less discomfort and with comparable pharmacokinetics to intramuscular administration [10]. This innovation has improved patient adherence and satisfaction, particularly among patients who prefer self-injection. Buccal mucosal systems, such as Striant, have provided yet another non-invasive option, delivering sustained-release testosterone through the oral mucosa [6].

The evolution of testosterone replacement therapy has been further refined through an understanding of individual variation in response and metabolism. Modern practice increasingly recognises the importance of individualising therapy based on patient preferences, comorbidities, and specific clinical contexts [11]. This includes careful attention to formulation choice in patients at risk for polycythemia—a recognised adverse effect of testosterone therapy. A comparative analysis has demonstrated that intranasal testosterone gel produces significantly lower increases in haematocrit than intramuscular testosterone cypionate, making it a potentially safer choice for patients with specific risk factors [12]. The recognition that different formulations produce different magnitudes of haematocrit elevation represents maturation in clinical pharmacology and personalised medicine approaches to testosterone therapy [13].

The modern era has also witnessed growing attention to microdosing strategies with testosterone cypionate and other formulations. Subcutaneous microdosing with testosterone cypionate involves the administration of small doses (10-50 mg) one to three times weekly, a departure from traditional larger doses given less frequently [10]. This approach aims to more closely approximate physiological testosterone production patterns, which typically show pulsatile secretion rather than constant levels. Proponents of microdosing argue it produces more stable serum levels, reduces haematocrit elevation, and may improve quality-of-life outcomes compared to standard dosing regimens. While formal clinical trials comparing microdosing to conventional protocols remain limited, accumulating clinical experience and small case series suggest potential benefits in terms of physiological approximation and adverse effect mitigation [10].

Recent pharmacological advances have also focused on selective androgen receptor modulators (SARMs) and other novel compounds designed to provide tissue-selective effects—stimulating anabolic effects in muscle and bone while minimising androgenic effects on prostate and sebaceous tissue [14]. Additionally, selective estrogen receptor modulators (SERMs) and aromatase inhibitors have been investigated as alternatives to direct testosterone replacement, particularly in younger men where preservation of fertility is paramount [14]. These emerging approaches represent the cutting edge of hormone replacement research, building on decades of fundamental understanding of testosterone physiology and pharmacology.

VII. Clinical integration and evidence-based monitoring (contemporary practice)

Modern testosterone replacement therapy benefits from extensive clinical trial data and established guidelines for patient selection, monitoring, and safety management [8]. The FDA’s 2014 warning regarding potential cardiovascular risks of testosterone therapy, followed by the 2025 statement clarifying that approved testosterone products did not increase risk of heart attack or stroke compared to placebo, reflects the maturation of clinical evidence and regulatory understanding [8]. Current clinical practice emphasises careful diagnosis of true hypogonadism through combined clinical and biochemical assessment, individualised therapy selection based on patient circumstances, and regular monitoring of adverse effects [15].

The therapeutic indications for testosterone replacement have been refined through clinical experience. Primary and secondary hypogonadism with documented low testosterone levels and clinical symptoms remain established indications [8]. However, the use of testosterone for age-related declines in testosterone without specific clinical hypogonadism remains controversial and is not universally approved for this indication [8]. Nevertheless, extensive clinical trials and meta-analyses have demonstrated that testosterone replacement therapy improves sexual function, quality of life, glycemic control, anaemia, bone density, and metabolic parameters in appropriately selected patients [16].

Summary

SummaryThis comprehensive timeline demonstrates how testosterone replacement therapy evolved from ancient empirical observation through surgical transplantation, chemical isolation and synthesis, to the sophisticated modern formulations and personalised approaches practised today. From Berthold’s pioneering demonstrations of internal secretion in 1849 to contemporary microdosing strategies with testosterone cypionate, the field has progressively refined both our understanding of testosterone physiology and our technological capacity to restore it safely and effectively. The trajectory reflects broader patterns in medicine: from observation to experimentation, from crude biological interventions to precise chemistry, and ultimately to individualised, evidence-based clinical practice tailored to each patient’s unique circumstances and needs.

Disclaimer: This article is for informational purposes only and is not a substitute for professional medical advice.

References:

[1] E. Nieschlag and S. Nieschlag, “Testosterone deficiency: a historical perspective,” Asian Journal of Andrology, Jan. 2014, doi: 10.4103/1008-682X.122358.

[2] F. H. A. Marshall, “Textbook of Endocrinology,” Nature, Jun. 1948, doi: 10.1038/161953a0.

[3] N. L. Miller and B. R. Fulmer, “Injection, Ligation and Transplantation: The Search for the Glandular Fountain of Youth,” Lippincott Williams & Wilkins, May 2007, https://www.auajournals.org/doi/10.1016/j.juro.2007.01.135

[4] G. F. Lydston, “Implantation of the Generative Glands and Its Therapeutic Possibilities,” Texas medical journal, Mar. 1915, [Online]. Available: https://www.semanticscholar.org/reader/1d173c3011e9bd0e5e7728998685457fbdb77868

[5] F. Lian and M. White, “FR02-09GEORGE FRANK LYDSTON: CONTROVERSIES IN VENEREAL DISEASES, TESTICULAR TRANSPLANTATION, AND THE AMERICAN EUGENICS MOVEMENT,” Journal of Urology, Apr. 2019, doi: 10.1097/01.JU.0000555422.46308.DB.

[6] M. Wald, R. B. Meacham, L. S. Ross, and C. Niederberger, “Testosterone Replacement Therapy for Older Men,” None, Feb. 2006, https://onlinelibrary.wiley.com/doi/10.2164/jandrol.05036

[7] K. Campbell, A. Muthigi, A. Ghomeshi, K. Schuppe, M. D. Sandler, and R. Ramasamy, “Safety Aspects and Rational Use of Testosterone Undecanoate in the Treatment of Testosterone Deficiency: Clinical Insights,” Dove Medical Press, Mar. 2023, https://www.dovepress.com/safety-aspects-and-rational-use-of-testosterone-undecanoate-in-the-tre-peer-reviewed-fulltext-article-DHPS

[8] E. Demir and E. Akpnar, “Testosterone Replacement Therapy: Indications, Application Protocols, and Side Effect Profile,” Recent Trends in Pharmacology, Jan. 2026, doi: 10.62425/rtpharma.1829150.

[9] U. Author, “In brief: Azmiro – a single-dose injectable formulation of testosterone cypionate.,” The Medical letter on drugs and therapeutics, Mar. 2025, doi: 10.58347/tml.2025.1724b.

[10] M. G. Figueiredo, T. GaglianoJuc, and S. Basaria, “Testosterone Therapy With Subcutaneous Injections: A Safe, Practical, and Reasonable Option,” Oxford University Press, Oct. 2021, https://academic.oup.com/jcem/article/107/3/614/6410585

[11] A. S. Dobs and K. J. Campbell, “An Individualized Approach to Managing Testosterone Therapy in the Primary Care Setting,” Dove Medical Press, Oct. 2022, https://www.dovepress.com/an-individualized-approach-to-managing-testosterone-therapy-in-the-pri-peer-reviewed-fulltext-article-IJGM

[12] J. C. Best, D. Gonzlez, T. A. Masterson, R. Blachman-Braun, R. Pai, and R. Ramasamy, “A cross-sectional comparison of secondary polycythemia in testosterone-deficient men treated with nasal testosterone gel vs. intramuscular testosterone cypionate,” None, Jul. 2020,https://cuaj.ca/index.php/journal/article/view/6651

[13] S. Nackeeran, T. Kohn, D. C. Gonzalez, J. White, J. Ory, and R. Ramasamy, “The Effect of Route of Testosterone on Changes in Hematocrit: A Systematic Review and Bayesian Network Meta-Analysis of Randomized Trials,” Journal of Urology, Aug. 2021

[14] V. A. Giagulli, A. Silvestrini, C. Bruno, V. Triggiani, A. Mordente, and A. Mancini, “Is There Room for SERMs or SARMs as Alternative Therapies for Adult Male Hypogonadism?,” Hindawi Publishing Corporation, Jan. 2020, https://onlinelibrary.wiley.com/doi/10.1155/2020/9649838

[15] E. V. Munari et al., “The complications of male hypogonadism: is it just a matter of low testosterone?,” Frontiers Media, Jun. 2023, https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2023.1201313/full

[16] N. Volkova, A. V. Safronenko, E. Gantsgorn, and Y. Degtyareva, “Clinical and pharmacological basis of the use of testosterone drugs for hormonal replacement therapy for hypogonadism in men,” Obesity and Metabolism, Aug. 2022,Fundamental differences