Lean Mass Gains, Cardiac Remodeling, and Muscle Oxidative Changes With High‐Dose Salbutamol During Resistance Training: A Randomized Controlled Trial

ABSTRACT

Misuse of beta₂‐agonists to promote leanness is increasingly reported among young men undertaking resistance training. However, preclinical models demonstrate adverse cardiac remodeling with high‐dose beta₂‐agonist. We investigated effects of prolonged supratherapeutic treatment with salbutamol on body composition, cardiac morphology and function, and muscle oxidative phenotyping during resistance training. In a double‐blind, randomized trial, 30 healthy, trained men (age 23 ± 2 years, mean ± SD) received oral salbutamol 16 mg/day (SAL) or placebo (PLA) during an 11‐week, supervised full‐body resistance training program (3 sessions/week). Assessments at baseline and follow‐up included dual‐energy X‐ray absorptiometry, echocardiography, cardiac magnetic resonance imaging (CMR), cardiopulmonary exercise testing (CPET), and vastus lateralis biopsies. The primary outcome was change in lean mass. Twenty‐six participants completed the study. Lean mass increased 1.8 kg more in SAL than PLA (95% CI 0.5–3.1; p < 0.01). CMR showed no between‐group differences in cardiac structure or function, including left ventricular mass. Echocardiography showed increased posterior, septal, and relative wall thickness in SAL compared to PLA (p < 0.01). Time to exhaustion during CPET did not change in SAL but increased by 7% in PLA (p < 0.01). Muscle capillary density and citrate synthase and 3‐hydroxyacyl‐CoA dehydrogenase activity decreased in SAL (all p < 0.01), with no such changes in PLA. While supratherapeutic salbutamol augmented lean mass gains during resistance training, it concurrently impaired muscle oxidative capacity, attenuated cardiopulmonary fitness improvements, and showed echocardiographic signals consistent with increased wall thickness. These risk–benefit trade‐offs inform illicit users of potential harms and support restrictions on supratherapeutic salbutamol in sports.

1. Introduction

While the main application of beta₂‐agonists is to treat bronchoconstriction, reports show beta₂‐agonists are increasingly abused by young men engaged in resistance training to promote leanness and muscle gains [1, 2, 3]. The prevalence of this practice is substantial. A cross‐sectional survey at four San Francisco gyms found that 10% and 22% of anabolic steroid abusers also used salbutamol and clenbuterol as part of their “supplements” for muscle gains [2]. Similarly, a web‐based survey of 500 respondents (99% male) recruited from online forums dedicated to anabolic agents revealed that 58% self‐reported clenbuterol use—a rate comparable to testosterone derivatives [3]. Beta₂‐agonists have a well‐documented repartitioning effect in animals and have been exploited as feed‐additives in livestock to increase meat production efficiency [4]. For example, salbutamol, the most commonly prescribed beta₂‐agonist, increased daily weight gain by 10% during a 7–8 week feeding period in pigs [5]. Similar, but smaller effects, have been reported in humans for which a few weeks of treatment with beta₂‐agonist promotes skeletal muscle growth [6, 7], which is related to increased myofibrillar protein synthesis and a net positive protein balance [8]. The doses used illicitly often exceed therapeutic levels by 4–8 fold, yet data at supratherapeutic doses during resistance training, which is the precise context of misuse, are lacking. This gap is particularly concerning given the rising prevalence of performance‐ and image‐enhancing drug use among young men in non‐competitive fitness settings [9].

Despite the high illicit use of salbutamol [1, 2], it is unclear whether prolonged abuse imposes detrimental effects on cardiopulmonary fitness, cardiac function, and muscle oxidative capacity. For many anabolic agents, adverse cardiac remodeling is a major concern [10, 11] and a problem not only pertaining to elite sport but also in the general population where use of anabolic agents is increasing [9, 12]. Although beta₂‐agonists are considered safe at inhaled therapeutic doses, prolonged supratherapeutic treatment can induce adverse cardiac geometric remodeling in rodents [13, 14] and case reports have linked beta₂‐agonist abuse with adverse cardiac events [15]. Preclinical data also point to skeletal muscle oxidative consequences. In pigs, dietary salbutamol reduced activities of muscle oxidative enzymes citrate synthase (CS) and 3‐hydroxyacyl‐CoA dehydrogenase (HADHA) [16]. And beta₂‐agonist–treated rodents exhibit impaired mitochondrial function and oxidative capacity [17, 18]. Collectively, these signals warrant human investigation to characterize potential risks, inform anti‐doping policy, and guide any consideration of expanding beta₂‐agonist indications beyond bronchodilation, for example, for muscle‐wasting conditions and weight loss, as previously suggested [7, 19, 20]. To date, no study has comprehensively characterized the integrated physiological impact of prolonged supratherapeutic salbutamol use in a resistance training context. While isolated effects on cardiac structure and muscle oxidative phenotype have been reported in preclinical models, the simultaneous assessment of whole‐body composition, cardiac remodeling, muscle oxidative phenotype, and cardiopulmonary fitness in humans has not been undertaken.

To comprehensively characterize the multi‐system effects of supratherapeutic beta₂‐agonist use, an integrated approach is required that simultaneously assesses anabolic outcomes, cardiac structure and function, skeletal muscle metabolic phenotype, and whole‐body cardiorespiratory capacity. Young men represent the primary demographic engaging in illicit beta₂‐agonist use within fitness settings [1, 2, 3], making them the relevant target population for risk characterization. Dual‐modality cardiac imaging combining echocardiography and cardiac magnetic resonance imaging (CMR) provides complementary diagnostic information. While echocardiography offers real‐time assessment and is typically adequate for evaluating hemodynamic parameters and valvular function, CMR provides superior volumetric quantification and more accurate measurement of myocardial thickness [21, 22]. Together, these modalities enable comprehensive cardiac evaluation and are essential for detecting early cardiac adaptations that may precede overt structural changes.

Therefore, we investigated whether prolonged daily use of salbutamol, at oral supratherapeutic doses (16 mg as a single dose), resembling those used in the resistance training milieu [6], would augment lean mass gains and induce cardiac remodeling in young men undergoing a period of resistance training. Furthermore, we examined adaptations in cardiac function, muscle oxidative capacity, capillarization, and cardiorespiratory fitness. We hypothesized that daily treatment with salbutamol would induce greater lean mass gains compared to placebo as well as induce left ventricular remodeling during a period of supervised resistance training.

2. Materials and Methods

2.1. Participants and Approvals

The study was performed at the Department of Nutrition, Exercise and Sports, University of Copenhagen and Departments of Cardiology and Respiratory Medicine, Bispebjerg‐Frederiksberg Hospitals. Participants were recruited from the Capital region area of Denmark through advertisements at the University campus and social networks. Inclusion criteria were healthy active young men, aged 18 to 40 years, oral and written informed consent, and a physically active lifestyle. Exclusion criteria were smoking, allergy towards the study drug, and chronic disease or use of prescription medication deemed by the investigators to interfere with the study outcomes. The study was conducted following the standards set by the 2013 version of the Declaration of Helsinki and was approved by the regional research ethics committee of the Capital Region, Denmark (H‐1‐2012‐119). The study was registered in ClinicalTrials.gov (NCT02551276).

2.2. Assessment of Eligibility

Before inclusion in the study, participants underwent a medical examination consisting of anamnesis and assessment of electrocardiography (ECG), lung and heart auscultation, and blood pressure measurement by a medical doctor. This was followed by measurement of body composition using Dual‐Energy X‐ray absorptiometry (DXA) (Lunar DPX‐iQ, Version 4.7, Lunar Corporation, Madison, WI, US).

2.3. Study Design and Randomization

The study was designed as a block‐randomized placebo‐controlled double‐blind parallel group trial with an experimental group (SAL) and a control group (PLA) for an 11‐week intervention with drug treatment and a standardized resistance training program (Figure 1A). Upon inclusion, participants were randomly allocated to the two groups (1:1 ratio), stratified for body mass. In SAL, participants ingested salbutamol (Ventolin, 4 mg tablets, GlaxoSmithKline, London, UK) in oral doses of 4 × 4 mg tablets daily as a single dose. In PLA, participants ingested 4 placebo tablets (lactose monohydrate/starch) daily as a single dose. Participants were told to refrain from competitive events for the entire study and not to change their daily physical activities and nutritional habits, which were recorded.

FIGURE 1.

Experimental workflow (A) and CONSORT diagram (B). CMR, cardiac magnetic resonance imaging; CPET, cardiopulmonary exercise test.

The primary outcome was change in lean body mass measured by DXA and the secondary outcome was change in LV‐mass measured by echocardiography. Other outcomes were markers of LV remodeling, muscle capillarization, and oxidative capacity, as well as absolute and relative V̇O_2max and exercise capacity. Tertiary outcomes pertaining to muscle calcium handling and fiber‐type composition have been reported elsewhere [23].

2.4. Study Drugs

Salbutamol is a commonly prescribed selective short‐acting beta₂‐agonist, which reaches peak systemic concentrations 1–3 h following oral ingestion [24]. We chose an oral daily dose of 16 mg as it induces a substantial beta₂‐adrenergic response in muscle [25] and resembles the regimen used within bodybuilding [6], which exceeds therapeutic doses of ~200 μg by inhalation which are normally prescribed to treat asthma symptoms. We considered an 11‐week intervention as sufficiently long to provide robust hypertrophy. The 11‐week intervention was selected based on established resistance training hypertrophy timelines and previous beta₂‐agonist trials demonstrating body composition changes within a few weeks [26, 27]. This timeframe also reflects realistic patterns of beta₂‐agonist cycling commonly reported in fitness settings [6]. Because chronic beta₂‐agonist treatment may induce tolerance [28], treatment was suspended for 4–6 days after the first four weeks of the intervention (re‐sensitization typically occurs after 3 days withdrawel [28]), after which treatment was reintroduced for another 6 weeks. Participants, investigators, and outcome assessors were blinded against treatment. The regional pharmacy of Copenhagen, Denmark, delivered salbutamol and placebo tablets. Randomization was conducted in SPSS (IBM, Armonk, NY, US) in blocks of five by staff not participating in the experimental procedures or data analysis. On training days, participants ingested the study drug 45 min prior to the training session under the supervision of study staff. This timing was chosen to ensure beta₂‐adrenergic receptor stimulation during the whole training session and to avoid potential attenuation of receptor responsiveness at peak systemic concentrations due to acute desensitization. On non‐training days, participants ingested the study drug between the hours of 08:00 and 22:00 and received electronic reminders. Drug compliance was assessed by diary records and counting the number of unused tablets in the bottle after the intervention compared with the number of tablets in the bottle before the intervention. The number of extra tablets in the bottle before the intervention was random.

2.5. Resistance Training

The resistance training consisted of a supervised progressive full‐body program (leg press, lunges, leg curls, bench‐press, row, lateral pull‐down, and military press) three times weekly. Target load of each set was 10 repetition maximum to failure with 1½ min recovery between sets. During the first week, participants performed two sets of each exercise, followed by three sets in week 2–7, four in week 8–9, and five in week 10–11. Between weeks 4 and 5, participants recovered for 4–6 days with no training. An instructor supervised every training session. Immediately after each training, participants drank a protein‐rich drink with carbohydrates (30 g whey protein, Arla Foods, Viby J, Denmark; 35 g carbohydrates, Maxim Sports Drink Orange, Orkla Care, Ishøj, Denmark) to stimulate post‐exercise protein synthesis. Total number of training sessions during the 11‐week intervention was 31. Participants were required to complete at least 28 training sessions (90%).

2.6. Pre‐ and Post‐Intervention Assessment

Before and after the intervention, participants attended two trials on separate days. During the first trial, we measured participants' whole body composition by DXA and cardiac morphology and function using two‐dimensional echocardiography. Hereafter, participants performed a standardized 10‐min warm‐up on a Monark E839 bike ergometer (Monark Exercise AB, Vansbro, Sweden) followed by a cardiopulmonary exercise test (CPET) to determine V̇O_2max and exercise capacity. The CPET started at 150 W, increasing by 30 W every minute until exhaustion and pulmonary gas exchange was measured breath‐by‐breath by indirect calorimetry (Oxycon CPX; CareFusion, San Diego, California, USA). V̇O_2max was defined as the highest average oxygen consumption over a consecutive 30‐s period and exercise capacity was defined as the time to exhaustion despite strong verbal encouragement. A CPET test was accepted as maximal either if a plateau in V̇O₂ was reached and maintained despite further increases in resistance, or a respiratory exchange ratio > 1.1.

During the second trial, participants underwent cardiac magnetic resonance imaging (CMR) followed by another DXA scan to reduce the effect of day‐to‐day variability. Furthermore, we sampled a muscle biopsy from the vastus lateralis of the right thigh using a Bergström needle with suction. Prior to biopsy sampling, an incision (3 mm) was made through the skin and fascia at the vastus lateralis belly under local anesthesia (2 mL lidocaine without epinephrine, 20 mg·mL⁻¹ Xylocain, AstraZeneca, Cambridge, UK).

Post‐testing trials were performed at least 36 h after the final day of treatment to ensure no residual action of salbutamol (duration of action 3–6 h).

Participants were told to abstain from caffeine, alcohol, nicotine, and strenuous exercise 48 h before each trial. To minimize variation, participants ingested a standardized meal and fluid 1½ h before trials.

2.7. Experimental Procedures

2.7.1. Dual‐Energy X‐Ray Absorptiometry

Participants were placed in the scanner in a supine position, undressed and euhydrated. Scans were preceded by 20 min of supine rest to allow distribution of body fluids. To reduce intra‐ and inter‐day variation, two scans were performed on two different days. Thus, each participant was scanned four times before and after the intervention and the mean was used for analysis. The scanner was calibrated before scans, using daily calibration procedures (Lunar “System Quality Assurance”). Scans were controlled for time of day and food intake leading up to the scan and were conducted by the same technician.

2.7.2. Two‐Dimensional Echocardiography

A transthoracic echocardiographic examination (TTE) was performed according to standard guidelines [29] (GE vivid E9 and GE Vivid S6, Vingmed, Horten, Norway). Expanded analysis of TTE raw data was performed according to current guidelines [22] (Analyzing software: GE Viewpoint cardiology V.6.12, EchoPAC suite V.204). Relative wall thickness (RWT) (RWT = LVPWD+IVSd/LVEDd) and left ventricular mass (LV_mass, g) were calculated according to Lang et al. [22]. The latter is indexed for body surface area (BSA) (LV_mass‐index, g/m²). BSA was calculated using the Du Bois formula. All echocardiographic analyses were performed by a single experienced investigator who was blinded to treatment allocation and time point. To assess measurement reliability, key parameters (including interventricular septal thickness, posterior wall thickness, left ventricular end‐diastolic diameter, and E/e′) were reanalyzed five times by the same investigator. The intra‐observer coefficient of variation for these measures was < 6%, indicating good reproducibility.

2.7.3. Cardiac Magnetic Resonance Imaging

CMR was performed using a 1.5‐Tesla Philips NT MRI scanner. Initial scout images were followed by cine steady‐state free precession breath‐hold, ECG‐gated sequences to capture two‐, three‐, four‐chamber, and short‐axis views. The short axis was identified using 2‐ and 4‐chamber images extending from the apex to (and including) the atria. Images were analyzed using the Circle Cardiovascular Imaging software, CVI42 (800 5 Ave SW #1100, Calgary, AB T2P 3T6, Canada). Short‐axis views were used for the calculation of volumes and masses. Endocardial and epicardial contours for end‐diastole and end‐systole were automatically traced and manually adjusted to calculate the LV end‐diastolic volume, end‐systolic volume, stroke volume, cardiac output, ejection fraction, and LV_mass. CMR images were analyzed offline by an experienced investigator blinded to group allocation and time point. Two approaches were tested for ventricular volumes and mass measurements. First, analyses were performed manually in accordance with standard guideline recommendations, excluding papillary muscles from left ventricular mass and including them in the blood pool. Second, an automated software‐based analysis was performed in which papillary muscles were included in left ventricular mass. Both analytical approaches yielded comparable results. Therefore, papillary muscles were included in the final calculation of LV_mass because the research question related to potential hypertrophic adaptations.

2.7.4. Muscle Biopsy Preparation

Sampled biopsies were divided in two pieces of which the first piece (20–30 mg) was mounted on an embedded medium (OCT Compound Tissue‐Tek; Sakura Finetek, Zoeterwoude, The Netherlands), frozen in isopentane cooled to the freezing point in liquid nitrogen, and stored at −80°C until immunohistochemical analysis. The other biopsy piece (50–100 mg) was washed in ice‐cold saline to reduce blood contamination, then dried and frozen in liquid nitrogen, and stored in cryo tubes at −80°C. Before immunoblotting and enzymatic activity assays each biopsy was freeze‐dried and dissected free from apparent non‐muscle tissue.

2.7.5. Immunohistochemistry and Confocal Imaging

Muscle fiber capillaries were identified by immunohistochemistry and confocal imaging as described previously [30]. In short, we cut embedded muscle samples in 8 μm transverse sections using a cryostat. Sections were fixed for 2 min in phosphate buffered saline (PBS, pH 7.2, Gibco 70013‐016, Life Technologies Denmark, Nærum, Denmark) containing 2% formaldehyde and washed in a 1:10 wash buffer (Dako S3006, Glostrup, Denmark), and blocked for 10 min in PBS containing 1% bovine serum albumin for immunohistochemical staining. Antibodies were diluted in antibody diluent (Dako S0809). Then, we visualized capillaries using biotinylated Ulex europaeus agglutinin I lectin (1:100; VECTB‐1065, VWR, Bie and Berntsen, Herlev, Denmark) and myofiber borders using a laminin antibody (1:500; Dako Z0097). Secondary antibodies [order listed: Streptavidin/FITC (1:200; DAKO F0422), Alexa‐555 donkey anti‐mouse (1:1000; Invitrogen A31570, Life Technologies Denmark), Alexa‐350 goat anti‐rabbit (1:1000; Invitrogen P10994) and Alexa‐488 donkey anti‐mouse (1:1000; Invitrogen A21202)] were used for the primary antibodies. Visualization was performed on a computer screen using a light microscope (Carl Zeiss, Germany), and all morphometric analyses were performed using a digital analysis program (ImageJ, NIH ImageJ). Two or more separate sections were used for analysis, and the cross‐sectional area was assessed by manually drawing the perimeter of each section. We then counted the number of muscle fibers and capillaries within each section. Capillary supply was subsequently expressed as capillaries per fiber (capillary‐to‐fiber ratio) and capillary density (cap·mm⁻²). The same blinded investigator performed all analyses.

2.7.6. Enzymatic Activity of Citrate Synthase and HADHA

Maximal enzyme activity of citrate synthase (CS) and HADHA was quantified in muscle homogenates using fluorometric methods (Fluoroscan Ascent, ThermoFisher Scientific) [31].

2.8. Statistical Analysis

Sample size was determined a priori for the primary endpoint (lean mass change). For a two‐group parallel design assuming α = 0.05, power = 80%, effect size of 1.4 kg based on Acheson et al. [26], and SD of 0.4 kg based on Fuller et al. [32], this required n = 13 per group. We recruited 30 participants to ensure adequate power accounting for potential dropouts. We used SPSS version 27 (IBM software, Armonk, NY, US) for subsequent statistical analyses. Data were normally distributed, as assessed using the Shapiro–Wilk's test and Q–Q plots, and are presented as means (±SD). To estimate within‐ and between‐group changes in SAL and PLA, we applied a two‐factor repeated measures linear mixed model with group and trial as fixed effects and participant as a random effect. Interaction effects were assessed for the primary endpoint and all secondary outcomes. Outcome statistics are presented as the mean delta effect size with 95% confidence intervals and p‐values to represent probability. An alpha ≤ 0.05 was considered statistically significant.

3. Results

3.1. Participants, Side Effects and Compliance

Of 34 participants screened, 30 fulfilled the eligibility criteria and were included in the study (Figure 1B), of whom 26 completed the intervention. Four participants withdrew due to training non‐compliance (n = 2 SAL; n = 2 PLA). In SAL, five participants reported sympathomimetic adverse events (muscle tremor, n = 4; palpitations, n = 3); all events were Grade 1, transient, and resolved without intervention. In PLA, three participants reported nausea (n = 3), all Grade 1. No Grade ≥ 2 adverse events were observed, and no participant discontinued due to adverse events. Estimated drug compliance rates were 89% for SAL and 92% for PLA; training compliance rates were 93% for SAL and 94% for PLA. Characteristics of the 26 completers are presented in Table 1. Participants were recreationally active prior to enrollment, engaging in team sports, running, biking, and/or light resistance training for 1–2 h weekly.

TABLE 1.

Participant characteristics.

	SAL (n = 13)	PLA (n = 13)
Anthropometrics and CPET
Age (years)	22 (±1)	23 (±3)
Height (cm)	186 (±5)	183 (±6)
Body weight (kg)	75 (±8)	76 (±9)
Lean mass (kg)	61.1 (±5.3)	59.3 (±4.6)
Fat mass (kg)	9.6 (±4.6)	11.4 (±6.9)
BSA (kg·m−2)	2.0 (±0.1)	2.0 (±0.1)
V̇O2max (L·min−1)	3.8 (±0.3)	3.7 (±0.4)
V̇O2max (mL·min−1·kg−1)	51.5 (±5.2)	48.6 (±5.8)
Exercise capacity (s)	456 (±59)	421
Echocardiography
Left ventricle and atrium morphology
IVSD (cm)	0.7 (±0.1)	0.8 (±0.1)
LVEDD (cm)	5.2 (±0.3)	4.9 (±0.3)
LVPWD (cm)	0.8 (±0.1)	0.9 (±0.1)
LVESD (cm)	3.4 (±0.4)	3.1 (±0.5)
RWT	0.29 (±0.03)	0.34 (±0.05)
LV‐mass index (g·BSA−1)	70 (±11)	71 (±14)
LA diameter (cm)	3.4 (±0.4)	3.2 (±0.4)
LA volume (mL) (4CH)	54 (±9)	52 (±8)
Systolic function
LVEDV (mL)	110 (±14)	102 (±16)
LVESV (mL)	44 (±12)	41 (±11)
LVEF (%), biplane	59 (±7)	61 (±6)
Fractional shortening (%)	35 (±5)	37 (±8)
Diastolic function
LV MV E vel (cm·s−1)	87 (±22)	93 (±16)
LV MV A vel (cm·s−1)	41 (±12)	39 (±7)
LV E/A	2.3 (±0.9)	2.5 (±0.5)
LV E/e′ lat	4.0 (±1.1)	4.4 (±0.9)
LV MV dec T (ms)	208 (±31)	186 (±37)

Note: Data are means (±SD).

Abbreviations: BSA, body surface area; CPET, cardiopulmonary exercise testing; LV, left ventricular; V̇O_2max, maximal oxygen consumption.

3.2. Body Composition

Resistance training led to large increases in lean mass for both groups, in which mass increased 1.8 kg (95% CI, 0.5 to 3.1; p = 0.009) more in SAL than PLA (Figure 2). Within‐group changes were 4.5 kg (95% CI, 3.7 to 5.4; p < 0.001) in SAL and 2.6 kg (95% CI, 1.7 to 3.7; p < 0.001) in PLA (Figure 2). Body mass increased proportionately with lean mass in both groups, while no apparent changes were observed in fat mass (Figure 2).

FIGURE 2.

Effect of 11 weeks of resistance training with daily oral ingestion of 16 mg salbutamol (SAL) or placebo (PLA) on whole body composition. Data are presented as mean change from baseline with the 95% confidence interval for SAL (red) and PLA (black). N = 13 for each group. **Within‐group change from baseline (p < 0.01). ^##Between‐group difference (p < 0.01).

3.3. Cardiac Geometric Remodeling

Echocardiography revealed geometric remodeling. LVmass‐index increased within SAL (Figure 3A; p = 0.002), though the between‐group difference versus PLA did not reach statistical significance (p = 0.14). In SAL, IVSD, LVPWT, and RWT increased by 14% (p = 0.007), 20% (p < 0.001), and 21% (p < 0.001), respectively, all significantly different from PLA (all p < 0.05) (Figure 3B,C). These changes occurred alongside a 3% reduction in LV_EDD (p = 0.020) in SAL, consistent with a concentric remodeling pattern. LA diameter and volume did not change significantly with the intervention in either group (Figure 3D).

FIGURE 3.

Effect of 11 weeks of resistance training with daily oral ingestion of 16 mg salbutamol (SAL) or placebo (PLA) on left ventricle (LV) and left atrium (LA) morphology and LV systolic and diastolic function assessed by echocardiography. (A) LV_mass‐index, (B) LV‐end diastolic diameter (LV_EDD) and LV‐end systolic diameter (LV_ESD), (C) Intraventricular septal wall diameter (IVSD) and LV posterior wall thickness (LV_PWT). (C) Relative wall thickness (RWT), (D) LA diameter and volume. (E) LV‐end diastolic volume (LV_EDV), LV‐end systolic volume (LV_ESV), LV‐ejection fraction (LV_EF), and fractional shortening. Data are presented as mean change from baseline with the 95% confidence interval for SAL (red squares) and PLA (black circles). N = 12–13 for each group. *Within‐group change from baseline (p < 0.05). **Within‐group change from baseline (p < 0.01). ^#Between‐group difference (p < 0.05). ^#Between‐group difference (p < 0.05). ^##Between‐group difference (p < 0.01).

CMR assessment showed no significant changes in LVmass‐index, LVEDV, or LVESV between groups (Table 2). LV_EF declined modestly across both groups (main effect of trial: p = 0.033), likely reflecting normal variation, with no between‐group difference (p = 0.597).

TABLE 2.

Cardiac magnetic resonance imaging before (PRE) and after (POST) prolonged daily treatment with salbutamol (SAL) or placebo (PLA) during 11 weeks of resistance training.

	SAL (n = 13)				PLA (n = 13)				Interaction
	PRE	POST	∆ (95% CI)	p	PRE	POST	∆ (95% CI)	p	∆ (95% CI)	p
LVEF (%)	62 ± 4	60 ± 6	−2 (−5 to 1)	0.13	64 ± 5	63 ± 5	−1 (−3 to 0)	0.09	−1 (−4 to 2)	0.60
Stroke volume (mL)	108 ± 18	107 ± 14	−1 (−9 to 7)	0.87	112 ± 14	105 ± 18	−7 (−13 to −1)	0.02*	6 (−3 to 16)	0.18
Cardiac output (L)	6.6 ± 1.1	6.8 ± 1.1	0.2 (−0.5 to 0.9)	0.54	6.8 ± 1.4	6.2 ± 1.1	−0.6 (−1.3 to 0.2)	0.12	0.8 (−0.2 to 1.8)	0.11
Heart rate (bpm)	62 ± 7	64 ± 7	2 (−3 to 6)	0.42	62 ± 11	61 ± 11	−1 (−8 to 6)	0.76	3 (−5 to 11)	0.47
LVEDV (mL)	174 ± 25	182 ± 25	8 (−4 to 19)	0.18	167 ± 18	164 ± 22	−3 (−9 to 3)	0.28	11 (−2 to 23)	0.09
LVESV (mL)	66 ± 9	74 ± 17	8 (0.0 to 16)	0.05*	59 ± 13	61 ± 14	2 (−1 to 5)	0.22	6.2 (−2.1 to 14.4)	0.13
LVmass‐index (g·BSA−1)	74 ± 11	74 ± 6	0 (−4 to 5)	0.91	83 ± 13	83 ± 16	0 (−3 to 3)	0.93	2 (−8 to 13)	0.89

Note: Data are means (±SD).

Abbreviations: EDV, end‐diastolic volume; EF, ejection fraction; ESV, end‐systolic volume; LV, left ventricular. *Within‐group change from baseline.

3.4. Systolic and Diastolic Function

Echocardiography assessment showed that LV_EDV, LV_ESV, and LV_EF did not change with the intervention in either group (all p > 0.05), whereas LV fractional shortening declined by 3.3%‐points in SAL only (95% CI, −0.2 to −6.4; p = 0.041) (Figure 3E).

CMR assessment showed an overall main effect of trial for LV_EF to decline by 1.8 percentage‐points (95% CI, 0.2 to 3.5; p = 0.033; Table 2) with no between‐group difference (p = 0.597). Stroke volume declined by 6% in PLA (p = 0.023) but was not significantly different from SAL (p = 0.183). LV_ESV increased in SAL (p = 0.049) but was not significantly different from PLA (p = 0.134). HR, LV_EDV, and LV_mass‐index did not change in either group (Table 2).

3.5. Maximal Oxygen Consumption and Exercise Capacity

When unadjusted for body mass, V̇O_2max did not change significantly with the intervention in either group, numerically increasing by 14 mL·min⁻¹ (95% CI, −118 to 146; p = 0.825) in SAL and by 95 mL·min⁻¹ (95% CI, −57 to 247; p = 0.199) in PLA. V̇O_2max relative to lean mass and time to exhaustion during the CPET changed differently between the groups during the intervention (p = 0.026 and p = 0.008, respectively), in which V̇O_2max relative to lean mass declined by 4.3 mL·min⁻¹·kg⁻¹ in SAL (95% CI, −2.5 to −6.0; p < 0.001), whereas no apparent change was observed in PLA (95% CI, −3.4 to 1.2 mL·min⁻¹·kg⁻¹; p = 0.324). Time to exhaustion during the CPET did not change in SAL (95% CI, −35 to 12 s; p = 0.305), while it increased by 7% with the intervention in PLA (95% CI, 9 to 53 s; p = 0.009).

3.6. Muscle Capillarization and Oxidative Enzyme Activity

Muscle capillary‐to‐fiber ratio increased by 17% (p < 0.001) and 16% (p < 0.001), respectively, with the intervention in SAL and PLA (Figure 4A), whereas capillary density declined by 8% (p = 0.005) in SAL but did not change significantly in PLA (Figure 4B,C). Muscle CS and HADHA maximal activity decreased by 10% (p = 0.01) and 15% (p < 0.001), respectively, in SAL, and differently from PLA (both p < 0.05) (Figure 4D).

FIGURE 4.

Effect of 11 weeks of resistance training with daily oral ingestion of 16 mg salbutamol (SAL) or placebo (PLA) on muscle capillarization and maximal enzyme activity of citrate synthase (CS) and hydroxyacyl‐CoA dehydrogenase (HADHA). (A) Number of capillaries per muscle fiber. (B) Number of capillaries relative to muscle fiber cross‐sectional area. (C) Representative confocal cross‐sectional image of capillaries (green) and muscle fiber borders (blue). (D) Maximal CS and HADHA enzyme activity. In left panels, bars represent the sample mean at baseline with error bars representing SD for SAL (red) and PLA (black). Right panels show the mean effect size with the 95% confidence interval for SAL (red squares) and PLA (black circles). N = 13 for each group. **Within‐group change from baseline (p < 0.01). ^#Between‐group difference (p < 0.05). ^##Between‐group difference (p < 0.01).

4. Discussion

This study provides the first comprehensive multi‐system assessment of supratherapeutic salbutamol effects in humans, integrating body composition, dual‐modality cardiac imaging, skeletal muscle oxidative phenotyping, and cardiopulmonary fitness testing. The major findings were: First, prolonged daily oral salbutamol at 16 mg markedly augmented whole‐body lean mass accrual during resistance training. Second, salbutamol suppressed skeletal muscle oxidative capacity, evidenced by reductions in maximal activities of oxidative enzymes. Third, salbutamol did not increase LV mass but was associated with subtle cardiac remodeling, with small increases in left ventricular posterior and septal wall thickness and higher relative wall thickness.

While the hypertrophic actions of beta₂‐agonists are well‐described [6, 7], the pronounced lean mass gain of 4.5 kg in SAL, corresponding to a weekly gain of around 400 g lean tissue, was notable and exceeds that reported in trials with other beta₂‐agonists. Prior work with the short‐acting beta₂‐agonist terbutaline reported lean mass gains of 1–2 kg over 4 weeks in healthy individuals, additive to resistance training [27, 33]. The lean mass gains induced by beta₂‐agonists appear to predominantly reflect accretion of additional myofibrils, rather than fluid retention or non‐muscle tissue expansion, as stable isotope techniques reveal apparent stimulation of myofibrillar protein synthesis as well as protein content accrual with beta₂‐agonist treatment [6, 7, 34]. In our study, lean mass increased by nearly 2 kg more in SAL than in PLA, underscoring the potency of salbutamol to augment lean mass gains and exposes why young men seeking particular body ideals can be tempted to illicitly use oral salbutamol as a performance‐ or image‐enhancing drug [1, 2, 15].

Our dual‐modality cardiac imaging revealed geometric remodeling in SAL without corresponding mass changes. Echocardiography detected significant increases in interventricular septal thickness (14%), posterior wall thickness (20%), and relative wall thickness (21%) in SAL versus PLA, while CMR showed no changes in LV_mass. The pattern of increased wall thickness preceding detectable mass changes indicates early‐stage concentric remodeling documented in longitudinal hypertension [35] and athletic training studies [36]. Importantly, resistance training alone (PLA) produced no cardiac changes despite 2.7 kg lean mass gain, indicating that SAL findings reflect direct beta₂‐adrenergic effects. Similar responses occur in beta₂‐agonist‐treated animals, with clenbuterol increasing wall thickness > 40% in horses [37] and inducing concentric hypertrophy in rodents [13]. Functionally, LV fractional shortening declined modestly in SAL (3.3 percentage points), though LV_EF remained unchanged by both modalities. This dissociation between structural and functional changes is typical of early remodeling where compensatory mechanisms maintain pump function.

We observed that salbutamol impaired indicators of cardiopulmonary fitness, including in exercise capacity and V̇O_2max relative to lean mass. The impairment in cardiopulmonary fitness likely reflects reduced capacity of the exercising muscles to extract and utilize oxygen. This is supported by the lower muscle capillary density and reductions in maximal activity of muscle oxidative enzymes CS and HADHA in SAL. With the absence of clear between‐group differences in LV volumes or ejection fraction, this pattern points to predominantly peripheral, rather than central, limitations to oxygen delivery and utilization. Given that we observed no apparent change in absolute V̇O_2max (i.e., unadjusted for body mass), the lower V̇O_2max relative to lean mass in SAL partly relates to muscle mass gains. But as highlighted by Habedank et al. [38], the net effect of beta₂‐agonist‐induced muscle mass gain is impaired peak V̇O₂ and power per kg active muscle mass during exhaustive exercise. A study with endurance trained individuals found no effect of a two‐week treatment period with 8 mg oral salbutamol on V̇O_2max [39]. This likely reflects the shorter duration of treatment and lower dose administered. Different types of beta₂‐agonists have been shown to impair V̇O_2max and aerobic exercise capacity in trained individuals [34]. Both terbutaline and salbutamol blunted the beneficial effects of endurance training in V̇O_2max and exercise capacity in healthy young individuals [30, 40]. In addition, daily treatment with terbutaline lowered V̇O_2max during a period of habitual lifestyle maintenance or resistance training [41]. From these findings we infer that prolonged supratherapeutic treatment with beta₂‐agonist compromises cardiorespiratory fitness.

The findings of the present study are relevant in the context of its misuse in resistance training settings [1, 2, 15]. While outcomes associated with long‐term abuse cannot be inferred from this 11‐week intervention, excessive beta₂‐agonist use has been linked to adverse cardiac events and cardiomyopathies in clinical reports [15] and high‐dose exposure induces cardiotoxicity in rodents [13, 14]. Although pharmacodynamics between beta₂‐agonists can differ [42], and hence their cardiac profile, clinical trials exploring beta₂‐agonists for muscle‐wasting conditions should prespecify and evaluate cardiac safety outcomes. From an anti‐doping perspective, our data support World Anti‐Doping Agency's (WADA) restrictions on systemic beta₂‐agonist use in and out of competition to secure athlete health. Most national anti‐doping and drug control programs currently emphasize clenbuterol screening in fitness centers. Our findings suggest that surveillance strategies could also include salbutamol.

Some limitations should be considered when interpreting our findings. First, echocardiography indicated small increases in left ventricular wall thickness with salbutamol, whereas CMR imaging did not detect corresponding changes in left ventricular mass or chamber volumes. Although CMR is considered the reference standard for assessment of ventricular volumes and mass, echocardiography remains widely used in clinical practice and may be more sensitive to dynamic or load‐dependent changes. The absence of structural remodeling on CMR suggests that the echocardiographic increase in wall thickness should be interpreted cautiously and may reflect methodological or physiological factors rather than true hypertrophic remodeling. Second, our study is slightly under powered (as revealed by post hoc power analysis) for detection of small changes in echocardiography, because the original sample size was calculated for the primary response variable (lean mass). Accordingly, while the statistically significant differences observed in wall thickness can partly be explained by insufficient power, the absence of changes in other echocardiographic or CMR‐derived structural measures should be interpreted with caution, as smaller between‐group differences may not have been detectable within the present sample size. Third, while we have not provided functional measures for strength gains (e.g., 1 repetition maximum), we refer to a separate investigation of this cohort in which we observed that participants treated with salbutamol exhibited greater increases in bike ergometer sprinting ability, but not in dynamic quadriceps muscle strenght [23].

In conclusion, our study demonstrates that daily treatment with salbutamol at supratherapeutic oral doses, resembling those used illicitly within resistance training settings, augmented lean mass gains during resistance training in healthy young men but concurrently compromised muscle oxidative capacity and cardiorespiratory fitness, while showing echocardiographic signs of increased wall thickness. These findings reveal a fundamental physiological trade‐off between anabolic and oxidative adaptations. Our results provide evidence‐based support for current WADA restrictions on systemic beta₂‐agonist use in sport, inform risk‐communication strategies targeting recreational users who may be unaware of cardiovascular and oxidative consequences, and underscore the need for rigorous cardiac and metabolic monitoring in any therapeutic trials of long‐term beta₂‐agonist use for muscle‐wasting conditions.

5. Perspectives

An important unanswered question is how long the observed cardiac and skeletal muscle changes persist following cessation of beta₂‐agonist exposure. As the present study was not designed or powered to assess reversibility, longitudinal follow‐up studies are warranted to determine the temporal dynamics of beta₂‐agonist–induced cardiac remodeling. Such work is particularly relevant to determine whether the observed adaptations reflect reversible, exposure‐dependent responses or more persistent remodeling.

Author Contributions

Morten Hostrup conceived the study. Morten Hostrup and Anders Kalsen designed the study. Morten Hostrup, Søren Jessen, Anders Kalsen, Michael Kreiberg, Vibeke Backer, and Jens Bangsbo conducted experimental trials pertaining to exercise testing, muscle biopsy sampling, and supervised the intervention. Susanne Glasius Tischer, Rakin Hadad, Philip Lykkebæk Bonde, Ahmad Sajadieh, and Hanne Rasmusen conducted echocardiography and CMR measurements and subsequent analyses. Anders Schulze Gad conducted the immunohistochemical analyses. Morten Hostrup wrote the first draft. All authors critically revised the manuscript and approved the final version of the manuscript.

Funding

The study was supported by an independent research grant from World Anti‐Doping Agency (Grant 11D5VB).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.