fifteen physically active and healthy volunteers ( four women ) , with at least 1 year 's experience in running exercise , participated in this study .
the volunteers , who were all at the same level of training , were instructed to maintain their regular exercise program until the 24 h period prior to the study , when they were instructed not to perform physical exercise .
all experimental procedures were performed in accordance with the ethical standards of the declaration of helsinki and were approved by the institutional ethics committee .
the volunteers were divided into two groups : one supplemented with l - arginine ( arg ) and the other with placebo ( pla ) by draw of lots ( simple randomization ) by a person outside the group of researchers participating in the study . on the day of the study , upon arriving at the training center , the volunteers remained at rest for 10 min .
once the blood samples were collected , volunteers were immediately given a supplement that consisted of a single oral dose of 12 capsules containing 500 mg of l - arginine hydrochloride or corn starch ( as placebo ) each ( total 6 g ) in 500 ml of deionized water .
thirty minutes after supplementation , the volunteers underwent two running exercise tests that consisted of 5 km time trial ( 5k - tt ) , meaning that the volunteers were instructed to run 5 km in the shortest time possible .
the recovery period between tests was 10 min and exercise performance was evaluated by measuring the trt . in addition
, blood samples were collected immediately after the first 5k - tt ( t1 ) , immediately after the second 5k - tt ( t2 ) , and after 20 min of rest ( t3 ) ( see fig .
the experimental procedures were performed in the period from 06:00 to 09:00 , and the volunteers were advised fast for at least 8 h before each visit .
the volunteers were not advised to control the ingestion of macronutrients ( i.e. protein , lipids , and carbohydrates ) from their diet on the day before the experimental procedures .
however , during this period , the volunteers did not consume any food , except water .
blood samples were collected in vacuum tubes containing separator gel and clot activator and centrifuged immediately at 3,000 g for 10 min at 4c to separate serum and were stored at 80c for later analysis .
the serum concentrations of insulin , gh , igf-1 , and cortisol were analyzed by enzyme immunoassay using commercially available kits ( abcan inc .
for all analyses , the reading was performed using a microplate reader ( victor x4 multilabel plate reader , perkin elmer , ca , usa ) .
the coefficient values of variation ( n=6 ) to insulin , gh , igf-1 , and cortisol were 4.9% , 3.7% , 4.4% , 7.5% for intra - assay and 7.2% , 8.4% , 9.1% , 12.8% for inter - assay , respectively .
two - way analysis of variance with repeated measures on two factors ( 24 ; grouptime ) was used to identify differences in variables , such as insulin , gh , igf-1 , and cortisol between the groups arg and pla and between time periods ( t0 , t1 , t2 , and t3 ) .
when a significant f was found , additional post hoc tests with bonferroni adjustment were performed .
furthermore , the statistical power ( 1- err prob ) was performed in order to detect the probability of type ii error occurring with regard to insulin , gh , igf-1 , cortisol , and trt in both 5k - tt tests .
all analyses were performed using a commercially available statistical package ( ibm spss statistics version 20 for windows , chicago , il , usa ) , and the results were expressed as meanssd .
fifteen physically active and healthy volunteers ( four women ) , with at least 1 year 's experience in running exercise , participated in this study .
the volunteers , who were all at the same level of training , were instructed to maintain their regular exercise program until the 24 h period prior to the study , when they were instructed not to perform physical exercise .
all experimental procedures were performed in accordance with the ethical standards of the declaration of helsinki and were approved by the institutional ethics committee .
the volunteers were divided into two groups : one supplemented with l - arginine ( arg ) and the other with placebo ( pla ) by draw of lots ( simple randomization ) by a person outside the group of researchers participating in the study . on the day of the study , upon arriving at the training center
once the blood samples were collected , volunteers were immediately given a supplement that consisted of a single oral dose of 12 capsules containing 500 mg of l - arginine hydrochloride or corn starch ( as placebo ) each ( total 6 g ) in 500 ml of deionized water .
thirty minutes after supplementation , the volunteers underwent two running exercise tests that consisted of 5 km time trial ( 5k - tt ) , meaning that the volunteers were instructed to run 5 km in the shortest time possible .
the recovery period between tests was 10 min and exercise performance was evaluated by measuring the trt .
in addition , blood samples were collected immediately after the first 5k - tt ( t1 ) , immediately after the second 5k - tt ( t2 ) , and after 20 min of rest ( t3 ) ( see fig .
the experimental procedures were performed in the period from 06:00 to 09:00 , and the volunteers were advised fast for at least 8 h before each visit .
the volunteers were not advised to control the ingestion of macronutrients ( i.e. protein , lipids , and carbohydrates ) from their diet on the day before the experimental procedures . however , during this period , the volunteers did not consume any food , except water .
blood samples were collected in vacuum tubes containing separator gel and clot activator and centrifuged immediately at 3,000 g for 10 min at 4c to separate serum and were stored at 80c for later analysis .
the serum concentrations of insulin , gh , igf-1 , and cortisol were analyzed by enzyme immunoassay using commercially available kits ( abcan inc .
for all analyses , the reading was performed using a microplate reader ( victor x4 multilabel plate reader , perkin elmer , ca , usa ) .
the coefficient values of variation ( n=6 ) to insulin , gh , igf-1 , and cortisol were 4.9% , 3.7% , 4.4% , 7.5% for intra - assay and 7.2% , 8.4% , 9.1% , 12.8% for inter - assay , respectively .
two - way analysis of variance with repeated measures on two factors ( 24 ; grouptime ) was used to identify differences in variables , such as insulin , gh , igf-1 , and cortisol between the groups arg and pla and between time periods ( t0 , t1 , t2 , and t3 ) .
when a significant f was found , additional post hoc tests with bonferroni adjustment were performed .
furthermore , the statistical power ( 1- err prob ) was performed in order to detect the probability of type ii error occurring with regard to insulin , gh , igf-1 , cortisol , and trt in both 5k - tt tests .
all analyses were performed using a commercially available statistical package ( ibm spss statistics version 20 for windows , chicago , il , usa ) , and the results were expressed as meanssd .
at baseline , no significant differences were observed with respect to age ( arg : 36.87.1 years vs. pla : 30.69.5 years ) , height ( arg : 172.310.6 cm vs. 175.07.5 cm ) , body mass ( 66.714.4 kg vs. 68.110.6 kg ) , and body mass index ( 22.22.6 kg / m vs. 22.12.2 kg / m ) .
the serum concentrations of insulin , gh , igf-1 , and cortisol during the study period are shown in fig .
both arg and pla groups showed a significant increase ( p < 0.05 ) in serum gh at all times ( t1 , t2 , and t3 ) , when compared to pre - supplementation ( t0 ) ( t0 : 3.280.95 ng / ml vs. 3.210.5 ng / ml ; t1 : 4.350.23 ng / ml vs. 4.170.13 ng / ml ; t2 : 4.220.25 ng / ml vs. 4.170.09 ng / ml ; t3 : 4.140.29 ng / ml vs. 4.130.18 ng / ml ) .
however , there were no significant differences among the conditions of supplementation ( arg vs. pla ) .
there was no significant differences between arg and pla in the serum concentrations of insulin ( t0 : 16.199.7 iu / ml vs. 17.688.28 iu / ml ; t1 : 14.736.84 iu / ml vs. 16.317.98 iu / ml ; t2 : 14.827.57 iu / ml vs. 15.038.78 iu / ml ; t3 : 13.416.45 iu / ml vs. 14.828.61 iu / ml ) and igf-1 ( t0 : 157.1173.32 ng / ml vs. 160.843.77 ng / ml ; t1 : 188.237.16 ng / ml vs. 175.1462.94 ng / ml ; t2 : 178.8543.16 ng / ml vs. 194.0859.13 ng / ml ; t3 : 195.2273.53 ng / ml vs. 182.1149.69 ng / ml ) over the time periods of the study .
serum cortisol showed no significant changes between the two groups ( arg vs. pla ) .
however , the pla group had a significant increase in the level of this hormone at t1 , t2 , and t3 compared to t0 , just as the concentration of cortisol in the arg group increased significantly at t1 and t2 compared to baseline measurement ( t0 ) , which was then followed by a significant reduction at t3 ( t0 : 198.7153.77 nmol / l vs. 207.5769.51
it is important to point out that the relevant statistical test in the present study had a virtually small power to detect the size differences between the arg and pla groups regarding : insulin ( 1- err prob=0.072 ) , gh ( 1- err prob=0.212 ) , igf-1 ( 1- err prob=0.367 ) , and cortisol ( 1- err prob=0.150 ) .
serum concentrations of insulin , growth hormone ( gh ) , insulin - like growth hormone 1 ( igf-1 ) , and cortisol before and after supplementation with l - arginine ( arg ) and placebo ( pla ) .
t0=baseline ; t1=immediately after the first exercise test ; t2=immediately after the second exercise test ; t3=after 20 min of rest ; ( a ) serum concentration of insulin ( iu / ml ) ; ( b ) serum concentration of gh ( ng / ml ) ; ( c ) serum concentration of cortisol ( nmol / l ) ; ( d ) serum concentration of igf-1 ( ng / ml ) . * denotes significantly different from t0 ( p<0.05 ) and   denotes significantly different from t1 and t2 ( p<0.05 ) .
the trt in the two sessions of 5k - tt in the arg and pla are depicted in fig .
3 . there was no significant difference in trt between the two tests , in both arg and pla ( first 5k - tt : 21:01:232:04:19 vs. 19:28:002:00:30 ; second 5k - tt : 21:24:072:28:28 vs. 20:01:302:23:32 ) .
the relevant statistical test in the present study had virtually a small power to detect a size difference between the arg and pla groups regarding trt in the two sessions of 5k - tt ( 1- err prob=0.059 ) .
total running time in the two sessions of a 5 km time - trial running test ( 5 km - tt ) after acute supplementation with l - arginine ( arg ) and placebo ( pla ) .
at baseline , no significant differences were observed with respect to age ( arg : 36.87.1 years vs. pla : 30.69.5 years ) , height ( arg : 172.310.6 cm vs. 175.07.5 cm ) , body mass ( 66.714.4 kg vs. 68.110.6 kg ) , and body mass index ( 22.22.6 kg / m vs. 22.12.2 kg / m ) .
the serum concentrations of insulin , gh , igf-1 , and cortisol during the study period are shown in fig .
2 . both arg and pla groups showed a significant increase ( p < 0.05 ) in serum gh at all times ( t1 , t2 , and t3 ) , when compared to pre - supplementation ( t0 ) ( t0 : 3.280.95 ng / ml vs. 3.210.5 ng / ml ; t1 : 4.350.23 ng / ml vs. 4.170.13 ng / ml ; t2 : 4.220.25 ng / ml vs. 4.170.09 ng / ml ; t3 : 4.140.29 ng / ml vs. 4.130.18 ng / ml ) .
however , there were no significant differences among the conditions of supplementation ( arg vs. pla ) .
there was no significant differences between arg and pla in the serum concentrations of insulin ( t0 : 16.199.7 iu / ml vs. 17.688.28 iu / ml ; t1 : 14.736.84 iu / ml vs. 16.317.98 iu / ml ; t2 : 14.827.57 iu / ml vs. 15.038.78 iu / ml ; t3 : 13.416.45 iu / ml vs. 14.828.61 iu / ml ) and igf-1 ( t0 : 157.1173.32 ng / ml vs. 160.843.77 ng / ml ; t1 : 188.237.16 ng / ml vs. 175.1462.94 ng / ml ; t2 : 178.8543.16 ng / ml vs. 194.0859.13 ng / ml ; t3 : 195.2273.53 ng / ml vs. 182.1149.69 ng / ml ) over the time periods of the study .
serum cortisol showed no significant changes between the two groups ( arg vs. pla ) .
however , the pla group had a significant increase in the level of this hormone at t1 , t2 , and t3 compared to t0 , just as the concentration of cortisol in the arg group increased significantly at t1 and t2 compared to baseline measurement ( t0 ) , which was then followed by a significant reduction at t3 ( t0 : 198.7153.77 nmol / l vs. 207.5769.51 nmol / l ; t1 :
458.16116.12 nmol / l vs. 433.26101.77 nmol / l ; t2 : 454.61125.21 nmol / l vs. 431.8874.82
it is important to point out that the relevant statistical test in the present study had a virtually small power to detect the size differences between the arg and pla groups regarding : insulin ( 1- err prob=0.072 ) , gh ( 1- err prob=0.212 ) , igf-1 ( 1- err prob=0.367 ) , and cortisol ( 1- err prob=0.150 ) .
serum concentrations of insulin , growth hormone ( gh ) , insulin - like growth hormone 1 ( igf-1 ) , and cortisol before and after supplementation with l - arginine ( arg ) and placebo ( pla ) .
t0=baseline ; t1=immediately after the first exercise test ; t2=immediately after the second exercise test ; t3=after 20 min of rest ; ( a ) serum concentration of insulin ( iu / ml ) ; ( b ) serum concentration of gh ( ng / ml ) ; ( c ) serum concentration of cortisol ( nmol / l ) ; ( d ) serum concentration of igf-1 ( ng / ml ) . * denotes significantly different from t0 ( p<0.05 ) and   denotes significantly different from t1 and t2 ( p<0.05 ) .
the trt in the two sessions of 5k - tt in the arg and pla are depicted in fig .
3 . there was no significant difference in trt between the two tests , in both arg and pla ( first 5k - tt : 21:01:232:04:19 vs. 19:28:002:00:30 ; second 5k - tt : 21:24:072:28:28 vs. 20:01:302:23:32 ) .
the relevant statistical test in the present study had virtually a small power to detect a size difference between the arg and pla groups regarding trt in the two sessions of 5k - tt ( 1- err prob=0.059 ) .
total running time in the two sessions of a 5 km time - trial running test ( 5 km - tt ) after acute supplementation with l - arginine ( arg ) and placebo ( pla ) .
this study evaluated the effects of 6 g of l - arginine supplementation on serum insulin , gh , igf-1 , and cortisol in response to running exercise in physically active individuals , as well as their performance during the running tests .
muscle glycogen synthesis can be optimized from increased skeletal muscle glucose uptake , which is enhanced by translocation of the glut-4 glucose transporters from intracellular vesicles to the cell membrane in response to insulin ( 7 ) .
( 4 ) demonstrated that systemic infusion of l - arginine ( 1 g min during 30 min ) in healthy subjects increased leg blood flow , which was associated with an increase in endogenous insulin .
however , during the present study , l - arginine supplementation did not affect insulin levels when compared to the placebo group over the time period of the study .
the supplementation dosage and the route of administration may have influenced the lack of significant changes in insulin levels after l - arginine supplementation .
previous studies have suggested that l - arginine administered orally does not reach systemic levels because almost 40% is catabolized in the small intestine ( 8 , 9 ) , which may be a possible explanation as to why some studies involving oral arginine supplementation have failed to show a significant change in the secretion of this hormone ( 5 , 8) .
there is evidence reporting exercise as a potent stimulator of gh secretion and explaining the mechanisms involved in this process ( 1013 ) .
furthermore , some studies have suggested that the amino acid l - arginine stimulates the secretion of gh by suppressing the secretion of somatostatin , a hormone that inhibits gh release ( 1417 ) .
these studies reported that l - arginine supplementation affects the exercise - induced gh response ; however , such results are controversial ( 5 , 6 , 17 , 18 ) .
the present study showed a significant increase in gh concentration in both arg and pla groups after running exercise , with no significant difference between the groups .
it appears that the response in gh release was mediated only by exercise , and that l - arginine supplementation induced no additional changes in this hormone when associated with running exercise .
( 19 ) who observed no significant increase in gh concentration after l - arginine and l - ornithine supplementation in nine trained athletes submitted to resistance training .
abel et al . ( 17 ) found no difference in plasma gh concentration after supplementation with arginine aspartate ( arg - asp ) in eight volunteers for 4 weeks with a low ( 5 g total  2.8 g of l - arginine and 2.2 g of aspartate ) and high ( 14.4 g total  5.7 g of l - arginine and 8.7 g of aspartate ) dose of these amino acids .
( 9 ) supplemented 14 physically active males with a low ( 0.075 g kg ) and high ( 0.15 g kg ) dose of orally administered l - arginine and found no significant change in serum gh . in the present study , no significant changes were observed in serum concentration of igf-1 after acute l - arginine supplementation .
forbes et al . , ( 8) supplemented physically active men with different doses of l - arginine ( 0.075 g kg or 0.15 g kg ) orally and observed no significant changes in serum igf-1 after 30 , 60 , 90 , 120 , and 180 min after supplementation .
other authors also found no significant changes in the serum concentration of igf-1 after oral l - arginine supplementation ( 6 , 20 ) .
cortisol is a catabolic hormone that increases during fasting or stress situations ( such as intense training ) ( 2124 ) .
we hypothesized that supplementation with l - arginine could reduce the catabolic effects of cortisol , since the secretion of anabolic hormones such as insulin and gh could act to offset the state of catabolism induced by cortisol . in the present study , there was a significant increase in serum cortisol immediately after two running exercise tests , when compared to presupplementation time ( t0 ) in both pla and arg groups .
therefore , it may be suggested that the response of cortisol secretion observed in the present study was only influenced by exercise .
the alleged effects of l - arginine supplementation on the release of anabolic hormones has stimulated athletes to use this amino acid for promoting greater gains in muscle mass , strength , and performance ( 12 ) . in the present study , there was no significant difference between groups ( arg and pla ) in the trt in the two sessions of 5k - tt . since we did not observe any significant effect from acute l - arginine supplementation on the hormonal parameters investigated in the present study ( e.g. increases in insulin or gh concentrations ) , no improvement in exercise performance
however , it is important to point out that with the very small sample size of 15 volunteers , the present study lacked sufficient power to detect any significant effect on serum concentrations of insulin , gh , igf-1 , and cortisol between the arg and pla groups , even if they exist in reality .
furthermore , because three of the four women who participated in the study were randomly allocated to the l - arginine group , and the other in the placebo group , no gender effect associated with the l - arginine supplementation was possible to determine .
in conclusion , it appears that oral l - arginine supplementation stimulates neither the production of insulin , gh , and igf-1 nor the reduction of cortisol .
based on the results of the present study , nutritional supplements based on l - arginine should not be recommended to induce hormonal changes and improve exercise performance in trained runners .
the research foundation of the state of rio de janeiro  faperj ( grant no . e-26/100.499/2012 and e-26/100.500/2012 ) provided financial support for the study .
 backgroundnutritional supplements based on the amino acid l - arginine have been hypothesized to improve exercise performance by increasing levels of insulin and growth hormone ( gh ) . 
 changes of these parameters in response to l - arginine supplementation may clarify the mechanisms underlying its putative physiological effects on physical performance.objectivethe aim of the study was to evaluate the effect of l - arginine supplementation on serum insulin , gh , growth factor insulin - like ( igf-1 ) , and cortisol in response to exercise . 
 exercise performance was also evaluated.designfifteen trained runners were divided into groups supplemented with 6 g of l - arginine ( arg ) or placebo ( pla ) . 
 blood samples were collected before supplementation ( t0 ) , immediately after the first exercise session ( t1 ) , after the second exercise session ( t2 ) , and after 20 min of rest ( t3 ) . the exercise consisted of two bouts of 5 km time - trial running 
 test.resultsthere was a significant increase in serum gh ( t0 : 3.280.95 vs. 3.210.5 ng / ml ; t1 : 4.350.23 vs. 4.170.13 ng / ml ; t2 : 4.220.25 vs. 4.170.09 ng / ml ; t3 : 4.140.29 vs. 4.130.18 ng / ml ) and cortisol ( t0 : 198.7153.77 vs. 207.5769.51 
 nmol / l ; t1 : 458.16116.12 vs. 433.26101.77 
 nmol / l ; t2 : 454.61125.21 vs. 431.8874.82 
 nmol / l ; t3 : 311.14102.91 vs. 362.26110.42 
 nmol / l ) after t1 , t2 , and t3 , with no significant difference between the arg and pla groups , respectively . 
 there was also no significant difference observed in the variables of igf-1 , insulin , and total running time between the arg and pla groups.conclusionsthe supplementation of l - arginine did not appear to stimulate the production of insulin , gh , and igf-1 and , thus , provided no benefit in hormonal response or exercise performance in trained runners . 
Materials and methods
Sample
Experimental design
Analysis of blood samples
Statistical analysis
Results
Baseline characteristics of volunteers
Hormonal parameters
Physical performance
Discussion
Conclusion
Conflict of interest and funding