Abstract
OBJECTIVE To determine the anabolic and lipolytic effects of a low dosage of clenbuterol administered orally in working and nonworking equids.
ANIMALS 8 nonworking horses and 47 polo ponies in active training.
PROCEDURES Each polo pony continued training and received either clenbuterol (0.8 μg/kg) or an equal volume of corn syrup (placebo) orally twice daily for 21 days, and then was evaluated for another 21-day period. Nonworking horses received clenbuterol or placebo at the same dosage for 21 days in a crossover trial (2 treatments/horse). For working and nonworking horses, percentage body fat (PBF) was estimated before treatment and then 2 and 3 times/wk, respectively. Body weight was measured at intervals.
RESULTS Full data sets were not available for 8 working horses. For working horses, a significant treatment effect of clenbuterol was detected by day 3 and continued through the last day of treatment; at day 21, the mean change in PBF from baseline following clenbuterol or placebo treatment was −0.80% (representing a 12% decrease in PBF) and −0.32%, respectively. By day 32 through 42 (without treatment), PBF change did not differ between groups. When treated with clenbuterol, the nonworking horses had a similar mean change in PBF from baseline from day 6 onward, which peaked at −0.75% on day 18 (an 8% decrease in PBF). Time and treatment had no significant effect on body weight in either experiment.
CONCLUSIONS AND CLINICAL RELEVANCE Among the study equids, long-term low-dose clenbuterol administration resulted in significant decreases in body fat with no loss in body weight.
Clenbuterol is an FDA-approved β2-adrenergic receptor agonist labeled for the treatment of recurrent airway obstruction in horses.1 The oral formulation has excellent bioavailability2 and accumulates in the lungs, liver, myocardium, and other tissues.3 Clenbuterol's main clinical use is as a bronchodilator,4,5 although it is also bronchoprotective against nonspecific inhaled triggers.6 However, β2-adrenergic receptors are located throughout the body, including in the heart, gastrointestinal tract, liver, uterus, blood vessels, sweat glands (in horses), fat, and skeletal muscles7; activation of these receptors by clenbuterol results in a panoply of physiologic effects. Clenbuterol has anti-inflammatory properties8,9 and increases the rate of mucociliary transport,10 which might have beneficial effects on the tracheobronchial clearance of inhaled particulates.11 When skeletal myocyte β2-adrenergic receptors are stimulated by clenbuterol, there is a direct anabolic effect12,13 that causes an increase in muscle mass in humans, food animals, and horses.14–16 In addition, clenbuterol induces lipolysis and inhibits adipogenesis17 associated with alterations in plasma concentrations of the adipokines leptin and adiponectin in adult horses.18 This increases muscle-directed protein deposition and reduces total body fat, which is generally known as repartitioning.14
Clenbuterol causes a switching from a type I, oxidative, slow-twitch fiber phenotype to a type II, glycolytic, fast-twitch fiber phenotype,19 even though β2-adrenergic receptors are expressed to a greater extent on slow-twitch than fast-twitch fibers.20 This effect is lessened when clenbuterol treatment is combined with exercise.21,22 Although these clenbuterol-induced changes in fiber type occur in horses, the contractile properties of isolated muscle fibers remain unchanged when treated with clenbuterol.23
Tachyphylaxis (functional tolerance) to many of the effects of β2-adrenergic receptor agonists is detectable after their administration for 7 to 14 days in both horses and humans.6,24 Aerobic exercise increases the density of β2-adrenergic receptors, thereby potentially minimizing expected tachyphylaxis to the repartitioning effects.20 Nonetheless, in a study of Standardbred mares,14 the anabolic effects of clenbuterol at a dosage of 2.4 μg/kg twice daily waned after 2 weeks, suggesting that tachyphylaxis to the effects on muscle and fat might also develop.
In light of these effects, clenbuterol has been investigated as a therapeutic compound to treat muscle wasting in humans,7 but because of concerns of abuse,25,26 its use in athletes has been banned by the International Olympic Committee27 and it is illegal to use it as a growth promoter in food animals.7 However, clenbuterol has long been a legal therapeutic medication, with published withdrawal times, in most horseracing jurisdictions. In several treadmill studies, clenbuterol has not been shown to improve horses' performance, and in fact, any changes noted have been negative ergogenic effects28–30 and deleterious effects on cardiac function.31 The anabolic effects of clenbuterol in horses have only been demonstrated at a dosage of 2.4 μg/kg twice daily.14 In the experience of the authors, most racehorse trainers and veterinarians do not typically administer this high a dosage; a dose of 5 mL (0.15 mg; approx 0.8 μg/kg) once or twice daily is commonly given to racehorses even in the absence of a specific diagnosis. Whether the repartitioning effects of clenbuterol, which have the potential to affect muscle mass and fat stores and thus potentially performance, occur at commonly used dosages, as opposed to those used predominantly in research settings, is of importance to regulatory bodies, trainers, and veterinarians.
The purpose of the study reported here was to determine the anabolic and lipolytic effects of a low dosage of clenbuterol administered orally in working and nonworking horses. The hypothesis was that clenbuterol, administered at the commonly used dosage of 0.8 μg/kg twice a day, would result in a reduction in PBF in working and nonworking equids and that exercise during treatment would affect the magnitude of this change.
Materials and Methods
A small group of nonworking horses and a larger group of polo ponies engaged in various levels of active polo training at 2 locations were used for the study. Two trials (involving the working and nonworking horses) were performed, and data were analyzed separately. These study protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee, and permission was gained from the owners or owners' agents for the horses' participation in the study.
Working horse protocol
Forty-seven client-owned, working polo ponies stabled at 2 facilities were randomly (paper lottery) assigned by location to receive clenbuterola (0.8 μg/kg, PO, q 12 h) or an equivalent volume of corn syrup (placebo) for 21 days. A 21-day washout period followed the placebo or drug administration, and investigators, owners, and trainers were blinded to the horses' group designation. There was no crossover in the design of this trial. Horses' designations were scheduled for 5 to 6 days of ridden exercise/wk including 2 to 10 chukkas (7-minute periods of polo competition) weekly during the study period. Given that these were client-owned, competing athletes, diet was determined by the barn managers and included hay and concentrate. Horses were weighed on a calibrated electronic scaleb before treatment commenced (baseline [day 0]), at day 21 (after final clenbuterol or placebo administration), and at day 42 (after 21 days of washout).
Nonworking horse protocol
Eight university-owned Thoroughbreds (7 females and 1 castrated male), aged 3 to 12 years old and weighing 536 to 615 kg, were assigned to a blinded, placebo-controlled trial. Because of the small group size of the nonworking horses, this trial had a crossover design. Horses were randomly (paper lottery) assigned to receive either clenbuterola (0.8 μg/kg, PO, q 12 h) at 7 am and 7 pm for 21 days or an equivalent volume of corn syrup (placebo) at 7 am and 7 pm for 21 days. Body weights were obtained before treatment commenced (baseline [day 0]), and then every 3 days with an electronic scaleb calibrated regularly as per manufacturer's recommendations. After a 7-week washout period, the experiment was repeated in a crossover fashion, allowing each horse to serve as its own control. The horses were maintained on goodquality hay and 10% to 12% protein feed; this diet was unchanged throughout each arm of the experiment. Horses were allowed access to a small paddock for 1 to 2 h/d and received no forced exercise. Body fat data for the nonworking horses were not collected during the washout period.
Body fat measurement
Estimation of body fat was performed in accordance with described techniques.32,33 In brief, a site 5 cm left lateral to the midpoint of the tuber sacrale was clipped of hair to ensure the same site was analyzed for subsequent measurements, and rump fat depth was measured by B-mode ultrasonography. Fat thickness measurements were obtained in triplicate in immediate succession by the same ultrasonographer with the same equipment for each study segment (nonworking horses, CABc; working horses, CMMd). For the nonworking horses, measurements were obtained every 3 days, and for the working horses, measurements were obtained twice per week. At each time point, fat thickness data for each horse were averaged, and the PBF was calculated from the mean by use of the following equation32:
For each horse at each time point, fat-free mass was obtained by multiplying the PBF by the body weight to obtain total fat mass, then subtracting this value from total body weight.14
Statistical analysis
At each time point, the PBF was subtracted from the baseline value to yield the change in PBF from baseline, which was analyzed as the primary outcome variable. The same approach was taken regarding fat-free mass to generate the change in fat-free mass from baseline for each time point after the baseline measurement. Data for the 2 trials (working and nonworking horses) were analyzed separately.
With a commercial software program,e data were checked for normality on the basis of a Tukey ladder. Mixed-effect modeling was performed to evaluate the effect of treatment (clenbuterol) on change in PBF from baseline and change in fat-free mass from baseline on the basis of time as a discrete covariate and horse as a random effect both within and between groups. Horses were grouped by their treatment (clenbuterol vs placebo), and the effects of age, sex, location (location 1 vs location 2), and breed were determined by Kruskal-Wallis analysis to compare values with nonparametric distributions. Values of P ≤ 0.05 were considered significant for all tests.
Results
Working horses
Thirty-nine of 47 working horses completed the full 42-day protocol. Horses exited the study due to sale or moving to different premises. The 39 horses for which data were available ranged in age from 6 to 22 years and weighed between 383 and 603 kg at the onset of the study period. Of the 39 included horses, 28 were Thoroughbreds, 2 were Thoroughbred crosses, and 9 were of mixed or unknown breed; there were 22 females, 16 castrated males, and 1 intact male. Twenty-three horses were at location 1, and 16 horses were at location 2. No complications associated with clenbuterol or placebo treatment were reported for any horse. Mean PBF at baseline for all working horses was 6.22% (range, 5.04% to 8.65%). At location 1, horses randomly assigned to receive clenbuterol (n = 11) had a mean baseline PBF of 6.07% (range, 5.12% to 6.84%) and a mean baseline weight of 477 kg (range, 445 to 525 kg), whereas those receiving placebo (n = 12) had a mean baseline PBF of 5.77% (range, 5.04% to 6.68%) and a mean baseline weight of 465 kg (range, 434 to 515 kg). At location 2, horses receiving clenbuterol (n = 8) had a mean baseline PBF of 6.81% (range, 5.42% to 7.76%) and a mean baseline weight of 495 kg (range, 421 to 603 kg), whereas those receiving placebo (n = 8) had a mean baseline PBF of 6.82% (range, 6.04% to 8.65%) and a mean baseline weight of 495 kg (range, 409 to 571 kg). Data were analyzed on an intent-to-treat basis, and 34 of the 39 horses played polo at least once a week throughout the study. There were no significant differences in percentage fat, fat-free mass, or weight at baseline between placebo-treated and clenbuterol-treated groups.
A significant (P = 0.04) effect in the clenbuterol-treated group was evident starting day 3 and was still increasing on the last day of clenbuterol treatment (Figure 1). Horses receiving clenbuterol had a mean ± SD change in PBF from baseline of −0.80 ± 0.54% at the end of the treatment period; horses receiving the placebo had a mean change in PBF from baseline of −0.32 ± 0.68%. This represented a peak decrease of 12% in PBF in the clenbuterol-treated group, which was significant (P < 0.001) both intra- and intergroup (ie, differing from baseline measurements and from the placebo-treated group over time). In the clenbuterol-treated group, there was a gradual return of PBF during the washout period (Figures 1 and 2); by day 32 (the 11th day of the washout period), there was no significant (P = 0.14) difference between the groups, which continued to the end of the study at day 42 (P = 0.25). Results were counterchecked by robust methods to ensure that outliers did not influence final results and were found to be equivalent. Time and treatment group had no significant (P > 0.05) effect on body weight at either location on days 0, 21, or 42.
Mean ± SD change in PBF from the baseline value (measured prior to treatment [day 0]) and at intervals through day 42 in working polo ponies administered clenbuterol (n = 19) or placebo (20). The data were pooled from horses at 2 locations. Each polo pony continued training and received either clenbuterol (0.8 μg/kg [solid line]) or an equal volume of corn syrup (placebo [dashed line]) orally twice daily for 21 days, and then was evaluated for a further 21-day period (washout period [shaded area]) when no treatments were given. Percentage body fat was derived on the basis of ultrasonographic rump fat measurements obtained lateral to the midpoint of the tuber sacrale. Fat thickness measurements were obtained twice a week in triplicate by the same ultrasonographer with the same equipment. At each time point, fat thickness data for each horse were averaged. *At this time point, values in the 2 groups differ significantly (P < 0.05). †At this time point, values in the 2 groups differ significantly (P < 0.01).
Citation: American Journal of Veterinary Research 76, 5; 10.2460/ajvr.76.5.460
Box-and-whisker plots of change in PBF from pretreatment baseline value at intervals through day 42 in the working polo ponies in Figure 1 at location 1 (A and B) and location 2 (C and D) that were administered placebo (A and C) or clenbuterol (B and D) for 21 days. In each panel, the shaded area indicates the 21-day washout period, when no medications were given. For each box, the lower and upper boundaries represent the 25th and 75th percentiles. The horizontal line within each box indicates the median values; whiskers represent the 10th (lower whiskers) and 90th (upper whiskers) percentiles. Observations outside these percentiles (outliers) are represented by dots.
Citation: American Journal of Veterinary Research 76, 5; 10.2460/ajvr.76.5.460
No significant (P = 0.20) treatment effect of clenbuterol on fat-free mass was observed after 21 days of clenbuterol administration (Figure 3), although the mean change in fat-free mass from baseline was −1.66 ± 22.90 kg in the clenbuterol-treated group, compared with a mean change in fat-free mass from baseline of 7.84 ± 10.72 kg in the placebo-treated group. At the end of the washout period (day 42), mean change in fat-free mass from baseline in horses receiving clenbuterol (mean ± SD change in fat-free mass from baseline, −7.27 ± 22.89 kg) had become closer in value to that of the horses receiving placebo (mean change in fat-free mass from baseline, −9.60 ± 9.96 kg). There was no significant (P = 0.85) difference in change in PBF from baseline by location. Sex (P = 0.59), age (P = 0.09), and breed (P = 0.42) were not different between treatment groups or locations.
Box-and-whisker plots of change in fat-free mass (FFM) from pretreatment baseline value (day 0) at days 21 and 42 in the working polo ponies in Figure 1 at location 1 (A and B) and location 2 (C and D) that were administered placebo (A and C) or clenbuterol (B and D) for 21 days. Fat-free mass was obtained by multiplying the PBF by the body weight to obtain total fat mass, then subtracting this value from total body weight. In each panel, the shaded area indicates the 21-day washout period, when no medications were given. For each location, no significant treatment effects of clenbuterol were observed at either time point. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 76, 5; 10.2460/ajvr.76.5.460
Nonworking horses
All 8 horses completed the full 21-day protocol on both occasions. At baseline, the nonworking horses in the clenbuterol and placebo treatment arms had mean PBF of 9.41% (range, 7.30% to 12.86%) and 9.14% (range, 7.30% to 11.18%), respectively, and mean weight of 565 kg (range, 532 to 615 kg) and 557 kg (range, 513 to 593 kg), respectively. Mean PBF and weight did not significantly differ between treatment arms.
When horses were treated with clenbuterol, a significant decrease in PBF from baseline was evident beginning on day 6 and continuing through day 18 (Figure 4). At day 6, the mean change in PBF from baseline was −0.35% (P = 0.04). Mean changes in PBF from baseline at days 9, 12, and 15 were −0.39% (P = 0.04), −0.54% (P = 0.01), and −0.49% (P = 0.03), respectively; at day 18, the greatest decrease from baseline of −0.75 ± 0.69% (P = 0.002) represented an 8% decrease in PBF. When horses were treated with placebo, there was a nonsignificant (P > 0.05) change in PBF from baseline of −0.40% at day 18. No significant (P = 0.49 to 0.68) effect of treatment on change in fat-free mass from baseline was evident at day 3, 6, 9, 12, 15, or 18; at day 18, clenbuterol or placebo treatment of the horses resulted in nonsignificant increases in fat-free mass (mean change in fat-free mass from baseline, 10.25 ± 17.76 kg and 6.88 ± 9.56, respectively [Figure 5]). No significant (P > 0.05) change in body weight was seen with time or treatment over the study period, and 2 of 4 horses that received clenbuterol in the first arm of the trial reverted to a higher baseline PBF by the beginning of the second arm of the trial after the washout period. Day 21 measurements were excluded owing to technical error. Overall, there was no significant (P = 0.47) difference in the maximum change in PBF from baseline between working and nonworking horses as a function of treatment.
Box-and-whisker plots of change in PBF from pretreatment baseline value (day 0) in 8 nonworking horses at intervals during treatment with placebo (A) or clenbuterol (B) for 21 days in a crossover trial (7-week interval between treatments). Data for day 21 were not available owing to technical error. *Within a treatment group, median value at this time point is significantly (P < 0.05) different from baseline. See Figures 2 and 3 for remainder of key.
Citation: American Journal of Veterinary Research 76, 5; 10.2460/ajvr.76.5.460
Box-and-whisker plots of change in fat-free mass (FFM) from pretreatment baseline value (day 0) in the 8 nonworking horses in Figure 4 that were administered placebo (A) or clenbuterol (B) for 21 days. No significant treatment effects of clenbuterol were observed at any time point. See Figures 2, 3, and 4 for remainder of key.
Citation: American Journal of Veterinary Research 76, 5; 10.2460/ajvr.76.5.460
Discussion
The purpose of the present study was to conduct double-blinded, placebo-controlled clinical trials to examine the interaction of exercise and long-term low-dosage clenbuterol (0.8 μg/kg, PO, q 12 h for 21 days) administration in adult horses. The results indicated that such long-term treatment caused a significant reduction in PBF from baseline of 8% and 12% in nonworking and working horses, respectively. There was no evidence of tachyphylaxis of the lipolytic effect by day 21 of treatment, and working horses returned to their baseline PBF within 2 weeks of cessation of clenbuterol treatment. Additionally, there was a smaller (albeit not significant) reduction in fat-free mass in working horses receiving clenbuterol than in working horses receiving placebo, and no significant change in body weight. These findings suggested that at this low dosage, clenbuterol reduces body fat (compared with the effect of a placebo) and may have effects that are both lipolytic and muscle-sparing in nature, although no evidence of anabolic effects that increased fat-free mass was observed during the 3-week treatment period.
Limitations of the present study included the imperfect method used for measuring PBF (rump fat measurements), although it has the advantage of being used in the previous study14 evaluating the anabolic effects of clenbuterol, thereby allowing fair comparison of present study data to previous findings, and has been used in a subsequent study.33 To make a clinically relevant assessment of the drug in a heterogeneous nonexperimental setting, no attempt was made to standardize other factors such as diet or exact amounts of exercise, given that effective randomization and placebo treatment groups were used to negate these confounders. Additionally, probably because the polo ponies started the study protocol in lean body condition, the absolute changes in rump fat depth were small, although consistently greater in those horses treated with clenbuterol. Likely attributable to the effect of exercise during the trial, the polo ponies receiving placebo also had a small diminution in PBF and fat-free mass over the course of the trial, although the fat loss was significantly less than that seen in polo ponies receiving clenbuterol. Although there was no increase in fat-free mass in the working horses treated with clenbuterol, a previous study14 did not reveal a positive effect of clenbuterol on fat-free mass in exercising horses until week 4 of administration. Furthermore, polo ponies do not undergo the same athletic demands as racehorses, but even low-goal (nonelite) polo ponies perform strenuous 7-minute chukkas at a level of exertion that results in prolonged heart rates > 75% of maximum.34 Finally, the 2 study protocols used were not identical, meaning that only very limited assessment of the interaction of exercise and clenbuterol was possible, and there was low statistical power due to high variability in the working horse group.
Horses competing at an elite level in multiple disciplines have a body composition of approximately 8% fat,28 similar to that of the polo ponies used in the present study, and higher fat-free mass is correlated with success in young Thoroughbred racehorses,35 endurance horses,36 and elite human athletes.37 In multiple species, high dosages of β2-adrenergic receptor agonists for 10 to 20 days have been shown to increase muscle mass by 10% to 25%.7 Considering that clenbuterol causes a repartitioning of fat to muscle, it would be intuitive that this drug would have performance-enhancing effects. Nonetheless, clenbuterol has consistently been found to have a negative ergogenic function in humans,25,38 laboratory animals,39 and equids.28–30 In experimental models, horses receiving high dosages of clenbuterol (2.4 μg/kg, q 12 h) have decreased aerobic capacity, time to exhaustion, and maximal oxygen consumption28 as well as ventricular remodeling with aortic enlargement, which was hypothesized to possibly increase the risk of fatal rupture.31
Although changes in muscle mass were not directly evaluated in the present study, the effects of clenbuterol on muscle tissue are considerable. Clenbuterol increases the type II-x, fast-twitch muscle fibers up to 70% in Standardbred mares, even when these horses are not exercising.40 On the basis of the suggestion that clenbuterol is effective mainly for improvement of muscle bulk rather than performance, it may be more likely to be abused with actual benefit by human bodybuilders26 and perhaps in equine halter horses or other disciplines where a callipygous phenotype is desirable. Despite published withdrawal times, there is no national standard for the withdrawal of clenbuterol in racehorses. It is identified as a class 3 therapeutic medication and has an accepted medical use in racehorses, which might have the ability to affect performance.1 In Pennsylvania, where the present study was performed, the racing commission currently requires a 48-hour withdrawal period prior to the start of a race when clenbuterol has been administered to a horse at a dose of 0.8 μg/kg.41
In adult horses that were and were not undergoing ridden exercise, a decrease in PBF with no change in body weight occurred in association with administration of clenbuterol at a dosage of 0.8 μg/kg twice daily, a third of a previously studied dosage. Although the anabolic and lipolytic effects of clenbuterol may cause fat and muscle repartitioning, the scientific literature does not support the conjecture that clenbuterol enhances performance with regard to aerobic exercise.
Acknowledgments
Supported by the Pennsylvania Horse and Harness Racing Commissions, Pennsylvania Harness Horsemen Association, Meadows Standardbred Owners Association, Pennsylvania Horsemen's Benevolent and Protective Association, and Pennsylvania Thoroughbred Horsemen's Association.
Presented in abstract form at the World Equine Airway Symposium with Veterinary Comparative Respiratory Society, Calgary, AB, Canada, July 2014, and the American College of Veterinary Internal Medicine Forum, Nashville, Tenn, June 2014.
The authors thank Hillary Goff, Alison Alessi, Morgan Agnew, Genevieve Comeau, and Courtney Pope for technical assistance.
ABBREVIATION
| PBF | Percentage body fat |
Footnotes
Ventipulmin, Boehringer Ingelheim, Ridgefield, Conn.
AL660 low profile large animal scale, Cambridge Scale Works Hone Brook, Pa.
Mylab 30 Vet, Esaote, Indianapolis, Ind.
EXAGO, Esaote, Indianapolis, Ind.
Stata, version 11.0, StataCorp, College Station, Tex.
References
1. Robinson NE. Clenbuterol and the horse, in Proceedings. 46th Annu Meet Am Assoc Equine Pract 2000; 229–233.
2. Soma LR, Uboh CE, Guan F, et al. Pharmacokinetics and disposition of clenbuterol in the horse. J Vet Pharmacol Ther 2004; 27: 71–77.
3. Soma LR, Uboh CE, Guan F, et al. Tissue distribution of clenbuterol in the horse. J Vet Pharmacol Ther 2004; 27: 91–98.
4. Erichsen DF, Aviad AD, Schultz RH, et al. Clinical efficacy and safety of clenbuterol HCl when administered to effect in horses with chronic obstructive pulmonary disease (COPD). Equine Vet J 1994; 26: 331–336.
5. Sasse HH, Hajer R. Veterinary and clinical experience of the use of a beta2-receptor-stimulating sympathicomimetic agent (NAB 365) in horses with respiratory disease [in Dutch]. Tijdschr Diergeneeskd 1977; 102: 1233–1238.
6. Read JR, Boston RC, Abraham G, et al. Effect of prolonged administration of clenbuterol on airway reactivity and sweating in horses with inflammatory airway disease. Am J Vet Res 2012; 73: 140–145.
7. Lynch GS, Ryall JG. Role of beta-adrenoceptor signaling in skeletal muscle: implications for muscle wasting and disease. Physiol Rev 2008; 88: 729–767.
8. Cudmore LA, Muurlink T, Whittem T, et al. Effects of oral clenbuterol on the clinical and inflammatory response to endotoxaemia in the horse. Res Vet Sci 2013; 94: 682–686.
9. Laan TT, Bull S, Pirie RS, et al. The anti-inflammatory effects of IV administered clenbuterol in horses with recurrent airway obstruction. Vet J 2006; 171: 429–437.
10. Turgut K, Sasse HH. Influence of clenbuterol on mucociliary transport in healthy horses and horses with chronic obstructive pulmonary disease. Vet Rec 1989; 125: 526–530.
11. Norton JL, Jackson K, Chen JW, et al. Effect of clenbuterol on tracheal mucociliary transport in horses undergoing simulated long-distance transportation. J Vet Intern Med 2013; 27: 1523–1527.
12. Hinkle RT, Hodge KM, Cody DB, et al. Skeletal muscle hypertrophy and anti-atrophy effects of clenbuterol are mediated by the β2-adrenergic receptor. Muscle Nerve 2002; 25: 729–734.
13. Navegantes LC, Resano NM, Migliorini RH, et al. Catecholamines inhibit Ca2+-dependent proteolysis in rat skeletal muscle through β2-adrenoceptors and cAMP. Am J Physiol Endocrinol Metab 2001; 281:E449–E454.
14. Kearns CF, McKeever KH, Malinowski K, et al. Chronic administration of therapeutic levels of clenbuterol acts as a repartitioning agent. J Appl Physiol 2001; 91: 2064–2070.
15. MacLennan PA, Edwards RHT. Effect of clenbuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates muscle β-adrenoceptors to induce hypertrophy. Biochem J 1989; 264: 573–579.
16. Claeys MC, Mulvaney DR, McCarthy FD, et al. Skeletal-muscle protein-synthesis and growth-hormone secretion in young lambs treated with clenbuterol. J Anim Sci 1989; 67: 2245–2254.
17. Kim HK, Della-Fera MA, Hausman DB, et al. Effect of clenbuterol on apoptosis, adipogenesis, and lipolysis in adipocytes. J Physiol Biochem 2010; 66: 197–203.
18. Kearns CF, McKeever KH, Malinowski K. Changes in adipopnectin, leptin, and fat mass after clenbuterol treatment in horses. Med Sci Sports Exerc 2006; 38: 262–267.
19. Gosker HR, Wouters EF, van der Vusse GJ, et al. Skeletal muscle dysfunction in chronic obstructive pulmonary disease and chronic heart failure: underlying mechanisms and therapy perspectives. Am J Clin Nutr 2000; 71: 1033–1047.
20. Williams RS, Caron MG, Daniel K. Skeletal-muscle beta-adrenergic receptors—variations due to fiber type and training. Am J Physiol 1984; 246:E160–E167.
21. Petrou M, Clarke S, Morrison K, et al. Clenbuterol increases stroke power and contractile speed of skeletal muscle for cardiac assist. Circulation 1999; 99: 713–720.
22. Hayes A, Williams DA. Contractile properties of clenbuterol-treated mdx muscle are enhanced by low-intensity swimming. J Appl Physiol (1985) 1997; 82: 435–439.
23. Plant DR, Kearns CF, McKeever KH, et al. Therapeutic clenbuterol treatment does not alter Ca2+ sensitivity of permeabilized fast muscle fibres from exercise trained or untrained horses. J Muscle Res Cell Motil 2003; 24: 471–476.
24. Haney S, Hancox RJ. Recovery from bronchoconstriction and bronchodilator tolerance. Clin Rev Allergy Immunol 2006; 31: 181–196.
25. Spann C, Winter ME. Effect of clenbuterol on athletic performance. Ann Pharmacother 1995; 29: 75–77.
26. Spiller HA, James KJ, Scholzen S, et al. A descriptive study of adverse events from clenbuterol misuse and abuse for weight loss and bodybuilding. Subst Abus 2013; 34: 306–312.
27. World Anti Doping Agency. 2014 Prohibited list international standard. Available at: www.wada-ama.org/Documents/World_Anti-Doping_Program/WADP-Prohibited-list/2014/WADA-prohibited-list-2014-EN.pdf. Accessed Jan 9, 2014.
28. Kearns CF, McKeever KH. Clenbuterol diminishes aerobic performance in horses. Med Sci Sports Exerc 2002; 34: 1976–1985.
29. Slocombe RF, Covelli G, Bayly WM. Respiratory mechanics of horses during stepwise treadmill exercise tests, and the effect of clenbuterol pretreatment on them. Aust Vet J 1992; 69: 221–225.
30. Rose RJ, Allen JR, Brock KA, et al. Effects of clenbuterol hydrochloride on certain respiratory and cardiovascular parameters in horses performing treadmill exercise. Res Vet Sci 1983; 35: 301–305.
31. Sleeper MM, Kearns CF, McKeever KH. Chronic clenbuterol administration negatively alters cardiac function. Med Sci Sports Exerc 2002; 34: 643–650.
32. Kane R, Fisher M, Parrett D, et al. Estimating fatness in horses, in Proceedings. 10th Equine Nutr Physiol Soc Symp 1987; 127–131.
33. Mazan MR, Deveney EF, DeWitt S, et al. Energetic cost of breathing, body composition, and pulmonary function in horses with recurrent airway obstruction. J Appl Physiol 2004; 97: 91–97.
34. Marlin DJ, Allen JC. Cardiovascular demands of competition on low-goal (non-elite) polo ponies. Equine Vet J 1999; 31: 378–382.
35. Fonseca RG, Kenny DA, Hill EW, et al. The relationship between body composition, training and race performance in a group of Thoroughbred flat racehorses. Equine Vet J 2013; 45: 552–557.
36. Lawrence L, Jackson S, Kline K, et al. Observations on body weight and condition of horses in a 150-mile endurance ride. J Equine Vet Sci 1992; 12: 320–324.
37. Thorland WG, Johnson GO, Cisar CJ, et al. Strength and anaerobic responses of elite young female sprint and distance runners. Med Sci Sports Exerc 1987; 19: 56–61.
38. Kamalakkannan G, Petrilli CM, George I, et al. Clenbuterol increases lean muscle mass but not endurance in patients with chronic heart failure. J Heart Lung Transplant 2008; 27: 457–461.
39. Duncan ND, Williams DA, Lynch GS. Deleterious effects of chronic clenbuterol treatment on endurance and sprint exercise performance in rats. Clin Sci (Lond) 2000; 98: 339–347.
40. Beekley MD, Ideus JM, Brechue WF, et al. Chronic clenbuterol administration alters myosin heavy chain composition in Standardbred mares. Vet J 2003; 165: 234–239.
41. Pennsylvania Racing Commission. Clenbuterol—suggested withdrawal time prior to race day. Administrative policy notice SHRC-2008–02. Harrisburg, Pa: Pennsylvania Department of Agriculture, 2008.
