Creatine and caffeine are among the main ergogenic agents used in sports aiming to achieve increased power, performance, lean body mass (LBM) and delayed fatigue [^1-5].

Creatine supplementation has been associated with increased LBM and strength [²,⁵,⁶] and reduced muscle mass loss [⁷]. Combined with power exercise, creatine supplementation may improve performance by spearing muscle glycogen, slowing down phosphocreatine dynamics in exercise and subsequent recovery and accelerating recovery between sets of exercise [^8-11], which subsequently may allow a greater number of exercise bouts to be performed. Thus, it may potentiate the strength exercise effects and result in increased LBM in humans and animals [¹¹,¹²]. However, its pharmacokinetics may by influenced by dietary components, such as caffeine and bicarbonate [¹³].

Caffeine ingestion enhances power output during high-intensity cycling in humans [¹⁴,¹⁵]. Caffeine is known to act directly on skeletal muscle leading to increased transmission of neural stimulus to the neuron-muscular junction [¹⁶]. It also blocks the central nervous system adenosine receptors [¹] and delay fatigue during power exercise in humans [¹⁶] and animals [¹,¹⁷]. These caffeine effects could enhance power training performance and hence promote alterations in body composition [¹⁸]. Nevertheless, the potential of chronic caffeine ingestion to enhance muscular strength and LBM has not been explored. Studies on the effects of acute caffeine ingestion on muscular strength have provided divergent data. For example, while a study by Jacobson et al. [¹⁹] demonstrated that a 7 mg/kg caffeine dose significantly enhanced muscular strength, Astorino et al. [²⁰] found no effect of a 6 mg/kg dose on humans.

Although a pre-workout supplement containing caffeine, creatine and amino acids combined with three weeks of high-intensity interval training increased the LBM in humans [²¹], the combined ingestion of creatine and caffeine may eliminate the ergogenic action of creatine supplementation, which is the increase in muscular stocks and exercise performance during intense intermittent exercise [¹³,²²,²³]. However, caffeine was found to be ergogenic when taken six days after creatine ingestion or caffeine abstinence [²⁴]. While creatine increased muscle phosphocreatine level and shortened muscle one-half relaxation time in rats [²⁵], short term caffeine intake inhibited muscle relaxation [²²]. This negative impact of caffeine on relaxation time contributes to counteract the beneficial effect of creatine supplementation on exercise training performance, which might affect the LBM composition.

Thus, the present study was carried to investigate the current uncertainties about the influence of creatine and caffeine associated with power exercise on the LBM composition and on the counteraction of these ergogenic agents. We also considered that the consumption of supplements in excessive doses might expose users to serious side effects [²⁶,²⁷], and that studies on human body composition are carried out using indirect measurements of the LBM [⁵,¹¹,²⁸,²⁹]. Thus, by using direct measurement of the LBM composition on a rat model, the purpose of this study was to determine whether high doses of caffeine and creatine supplementation, either solely or combined, affect the LBM composition of rats submitted to a power training regime based on a model of intermittent vertical jumps.

The concentrations of blood lactate increased similarly in all exercised animals (ANOVA One-Way Repeated Measures, P < 0.05) from rest (2.7±0.6 mmol/L; mean ± SD), to the second set (6.9 ± 1.4 mmol/L) and fourth set (9.2 ± 1.8 mmol/L) of vertical jumping moments.

We demonstrated that supplementation with high combined doses of creatine and caffeine did not affect the LBM composition of either sedentary or exercised rats. However, caffeine supplementation alone reduced the percentage of fat in the carcass. In addition, the employed model of power training increased the percentages of water and protein and reduced the fat percentage in rats.

One of the main observations of our study was that animals supplemented with creatine or creatine plus caffeine did not present increased water retention in skeletal muscles (carcass). It is suggested that creatine supplementation leads to intramuscular water accumulation caused by its high osmotic power [⁷,³³]. Our results do not corroborate such hypothesis and are consistent with the similarity of body weight among our experimental groups as an increase in water retention in response to creatine ingestion might have augmented body weight [¹³,³⁴]. Even though the methods of weighing were indirect, this lack of increase in body weight caused by creatine supplementation has been reported elsewhere [²,¹¹,²⁹].

Despite the fact that caffeine exerts a slight diuretic effect [¹⁵], which could have reduced water content, contrasting the effects of creatine [³⁵], our study revealed that caffeine ingestion did not affect the percentage of water in the lean body mass. Similar results were found by Vanakoski et al. [³⁶], although in our experiment, caffeine dosage was 2.14 times higher.

Concerning exercise effects, we observed an increased percentage of water in the carcass of the exercised animals. Although we have not assessed the content of muscle glycogen, it is thought that such effect is associated with the ability of exercise to promote accumulation of muscular glycogen, since 2.7 g of water are incorporated in the muscle per each gram of glycogen incorporated [³⁷]. Our results agree with those reported by Cortright et al. [³⁸].

Our observation that creatine or creatine plus caffeine did not affect the protein percentage of lean body mass demonstrates the absence of differences in body weight among our experimental groups. The increased in muscular mass in response to creatine ingestion [⁵,¹¹,³⁴,³⁵] may be due to the increased water retention in muscles [¹³]. However, such effect was not observed in the present study. Similar results were found by others [²,²⁹,³⁹]. When measuring urinary nitrogen, Jowko et al. [⁴⁰] verified that creatine did not affect the retention of body nitrogen, suggesting that creatine would not increase protein incorporation. In the present study, even though creatine doses were very high, it did not affect protein percentages in the carcass of creatine supplemented groups.

We demonstrated that the exercise training regime employed here increased the percentage of protein in the carcass, despite the reduction in the final body weight. This finding is consistent with those presented in the literature inasmuch as there is a large body of evidences of skeletal and cardiac muscle hypertrophy in response to intermittent power and running exercises in humans and animals [¹¹,¹²,⁴⁰].

Our results revealed that high-dose caffeine supplementation reduced the fat percentage of the lean body mass as compared to creatine ingestion, independently of the exercise training. It has not been mentioned the direct effect of creatine on skeletal muscle fat [²,¹¹]. However, the ingestion of caffeine may increase the turnover and mobilization of free fatty acid [²²,⁴¹,⁴²] and save muscular glycogen storages [²²], which would result in reduced body weight [⁴²]. Caffeine intake increases the basal metabolic rate and catecholamine release [⁴¹,⁴³]. Caffeine may also inhibit the activity of the phosphodiesterase enzyme, which increases the levels of AMPc and reduces the activity of hormone-sensitive lipase, leading to higher lipolysis [⁴⁴]. However, we found no differences in body weight among the groups SPl and EPl, as compared to SCaf and ECaf, respectively. We also demonstrated that the group SCaf presented higher body weight than ECaf and that the exercised animals exhibited lower body weight, as compared to the sedentary animals. Therefore, such reduction in the percentage of fat in the carcass of animals supplemented with caffeine may indicate the interference of exercise instead of caffeine ingestion.

We observed that the exercised animals exhibited lower body weight as well as lower fat percentages compared to the sedentary animals. Although in our model of power exercise the main source of energy is the anaerobic glycolysis, oxygen consumption continues high after exercise due to the increased energetic metabolism of active muscles, an effect of post-exercise oxygen consumption (EPOC) [²⁸,⁴⁵]. Therefore, such reduction in fat percentage might have not been caused by energy consumption during the vertical jump sets, but partly by oxygen deficit and post-exercise energy costs via EPOC. Malatesta et al. [²⁸] demonstrated that lipid oxidation during post exercise recovery increased in response to intermittent and continuous exercise compared with the time-matched no-exercise controls. The lower fat percentage in the lean body mass of the exercised animals might have resulted from their higher protein percentage as compared to the sedentary animals.

In the present study, neither supplementations nor exercise training affected the excretion of urinary creatinine during the first week. In the second week, the creatinine from the groups creatine or creatine plus caffeine was higher than that from the placebo group, and also higher as compared to the first week. On the other hand, urinary creatinine decreased. Thus, the significance of creatine and creatine plus caffeine effects from the second week has disappeared. These results indicate that the ingestion of high doses of creatine (0.431 g·kg) during the load phase promoted increased excretion of urinary creatinine via a non-enzymatic reaction, as demonstrated by other authors [¹³,²⁹,⁴⁵]. Our data also suggest that the load phase could be more important in increasing body creatine storages, since after the phase of creatine maintenance (6^th week), urinary creatinine excretion was reduced.

Finally, caffeine ingestion did not affect creatinine excretion. Such finding suggests that caffeine ingestion had no effect on creatine pharmacokinetics. However, our data do not allow us to substantiate such suggestion as we did not measure the muscular content of creatine and its clearance. This is a limitation of this study and requests further investigations.

In conclusion, high combined doses of creatine and caffeine does not affect the LBM composition of either sedentary or exercised rats, however, caffeine supplementation alone reduces the percentage of fat in the carcass. The employed vertical jump regimen increases the percentages of water and protein and reduces the fat percentage in these animals.

The authors declare that they have no competing interests.

All authors have read and approved the final manuscript. AJN is the principal investigator of the project. FSCF, NMBC and AJN designed the study; FSCF, SAF and MACJ collected the data; FSCF and AJN conducted data analysis; FSCF and AJN wrote the manuscript.

The authors wish to thank BIOCLIN^® Laboratory for the calcium and creatinine analysis kits. This study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG (CDS 973/2004). FSCF held a scholarship from CAPES (PIQDTEC 320.440.1-1). AJN is a CNPq fellow.