cortisol is principally glucocorticoid secreted from the adrenal cortex into human serum or 
 saliva and plays a major role in metabolism , the immune function , and the regulation of 
 physiological stress responses1 .
a strong 
 association has been reported between salivary cortisol and blood sample ratios which 
 provides an accurate assessment of unbound cortisol levels2 , 3 .
stress is explained as physiological changes occurring in the human body in relation to 
 physical or external stress conditions which drastically and negatively affect the 
 homeostasis4,5,6 , and differences in 
 psychological and physical stressors resulting in hormonal changes7 , 8 .
it has been reported that accommodation of psychological and physical stressors mainly 
 depends on the activity of the adrenal glands .
bodies under stress need higher rates of 
 metabolic processing along with glucocorticoid release which may affect physical 
 activity9,10,11 .
physically active people of different ages show better emotional health , and it has been 
 found that regular physical activity increases the fitness of persons and their ability to 
 conquer depressive mood12 .
the change of 
 endocrine hormones among individuals exposed to different types of stress has been 
 investigated . a significant increase in the cortisol level ,
a marker of endocrine response 
 was reported in subjects stressed by high intensity physical activity9 .
also , it was reported that participants with low physical 
 activity showed larger cortisol responses to physical exercise than those participated 
 regular exercise13 , and that regular 
 exercise modulates the response of cortisol elevation over time9 .
. decreased levels of cortisol were reported in individuals performing both low 
 intensity14 , and acute exercise 
 training15 , 16 .
many exercise interventions are designed to manipulate the mechanistic effects of physical 
 activity such as behavioral and physiological actions on cortisol levels .
recent studies 
 have reported that the adaptation of endocrine system to exercise training occurs 
 physiologically via buffering of anabolic and catabolic processes17 .
for example , the change in the cortisol / testosterone 
 ratio , as an indicator of the anabolic - catabolic balance , has been used with limited success 
 to determine the physiological strain of exercise training18 .
the free - testosterone / cortisol ratio ( fr - t / c ) has been proposed as 
 a major indicator of anabolic and catabolic effects due to over training especially among 
 athletes . it was reported that , a reduction in the testosterone / cortisol ratio of more than 
 30% is related to overtraining status19 . 

also , in another study , there was a noticeable increase in the levels of testosterone 
 without a big difference in cortisol levels after maximum aerobic training20 , and this variation in adrenal hormones 
 may be related to subjects having differences in their response to exercise21 . while , cortisol has a catabolic effect , 
 testosterone is responsible for the stimulation of the anabolic process of skeletal muscle 
 growth which increases linearly in response to exercise22 .
the increase of cytosolic proteins in the circulation including lactate dehydrogenase ( ldh ) 
 after exercise reflects muscular injury whereas both exercise intensity and duration 
 independently affect enzymatic activity and muscular pain with intensity being the variable 
 with the greatest effect23 , 24 .
it has also been reported that adjusting lactate levels 
 plays an integral part in overtraining prevention , since it is an accessible and widely used 
 technique of overtraining diagnosis in the sports field25 .
many previous studies have investigated changes in cortisol and testosterone hormones in 
 response to exercise training and the differences in adaptation to exercise among subjects 
 however ; their results are contradictory regarding the intensity and type of exercise26,27,28 . both cortisol and testosterone show significant linearity with peak concentrations in 
 response to exercise once a specific intensity threshold is reached29 , even under low intensity exercise with a long enough 
 duration30 , 31 .
previous , studies have reported the change in stress hormones at 
 different durations of training programs of varying intensities32,33,34,35 , their results 
 suggest that salivary stress hormones are a potentially valid outcome measures of responses 
 to exercise training programs . while , there are many studies on the effects of short 
 training periods on stress hormones36 , 37 , little is known about the effect of short 
 term exercise of moderate intensity on the status of salivary stress hormones even though it 
 may be of use in diseases related to older adults such as cognitive impairment and 
 cardiovascular diseases38 , 39
. therefore , the present study assessed the effects of 4 
 weeks of moderate aerobic exercise on saliva stress hormones and lactate levels in healthy 
 adult volunteers because short term exercise of moderate intensity may be suitable for most 
 sedentary and elderly subjects
sixteen healthy students with ages between 15 and 25  years were recruited for this study 
 ( table 1table 1.anthropometric characteristics of the participants ( mean  sd)no.anthropometricparametersparticipants(no.= 16 ; mean  sd)1.age ( years)22.82.142.height ( cm)172.0  4.923.weight ( kg)69.5  3.74.bmi ( kg / m)23.7  1.41 ) .
none of the participants were involved in any specific training program or 
 normal physical activity during the study schedule .
subjects with a bmi of more than 25 
 ( kg / m ) , ischemic heart disease , or with severe orthopedic problems were 
 excluded from this study .
prior to the exercise test , the risks and benefits of the study 
 were explained , and written informed consent was obtained from each participant after a 
 medical check - up to ensure that they were fit , healthy and had no physical limitations .
all 
 participants were advised to keep to their normal habits of eating during the entire period 
 of data collection .
the experiment was conducted in accordance with the ethical guidelines 
 of the 1975 declaration of helsinki , and was reviewed and approved by ethical committee of 
 the rehabilitation research chair ( rrc ) , king saud university , riyadh , ksa .
each subject participated in an exercise training program three times per week for 4 weeks . 
 the exercise program consisted of treadmill walking .
each individual s training intensity 
 was calculated as the training heart rate ( thr ) based on the subject s age and predicted 
 maximum heart rate and resting heart rate according to karvonen s formula40 , ( thr = hrrest + ( hrmax  hrrest )  tf , 
 where hrrest = resting heart rate in bpm .
hrmax = maximum heart rate in bpm and tf = 
 training fraction ( 65 to 75% for the moderate intensity used in this study ) .
this formula 
 has previously been shown to be valid for the effect of exercise on stress hormones41 , 42 .
each exercise session consisted of three phases ; the warm - up , 
 active , and cool - down phases . during the warm - up phase , the subjects performed simple 
 stretching exercise for all large muscle groups and walked for 5 to 10 minutes at tf equal 
 to 3040% . during the active phase , the subjects were encouraged to reach their 
 pre - calculated training heart rate ( thr ) in bouts with a total time of 30 to 45 minutes .
the 
 last phase was cool - down which continued for 10 to 15 minutes during which the workload 
 gradually decreased until hr and blood pressure had nearly returned to their resting levels . 
 throughout the entire training session ,
the subjects were monitored by a portable heart rate 
 monitor to keep the exercise intensity within the precalculated training heart rate for each 
 subject .
samples of saliva were taken from the participants during a rest day ( 24 hours after 
 training ) in the mid - morning ( 9:0011:30 am ) , following overnight fasting .
samples were 
 separated using centrifuge , numbered , and then stored at 80c until used for analysis . after the saliva had been separated , the lactate levels were measured by the uv method , 
 provided with the caymans l - lactate assay kit ( cat no 700510 , usa ) .
cortisol and 
 testosterone levels ( pg / ml ) were measured in the saliva samples of participants using the 
 immunoassay technique according to the instructions of the cortisol elisa - kit ( diagnostics 
 biochem canada , inc . ) and testosterone elisa - kit ( cat no .
repeated measures analysis of variance ( anova ) was used to analyze the effect of the 
 exercise on the levels of the studied stress hormones . the statistical relation among 
 hormone levels pre- and post - exercise
were calculated using pearson coefficients , and the 
 significance of differences between the mean values before and after each exercise session 
 were tested using the paired samples t - tests .
the effect of short term exercise on the levels of stress hormones was assessed after 4 
 weeks of training .
the participants were recommended to keep to their normal routine diet 
 throughout the study to avoid dietary effects on the measured variables .
the data of stress 
 hormones in the pre- and post - training recovery stage are shown in table 2table 2.differences between pre- and post - training concentrations of selected 
 biomarkersvariablespre - trainingvalue(mean  sd)post- training value(mean  sd)mean differencecortisol [ c ] ( pg / ml)17.51  3.1837.2  4.1719.71  4.66**testosterone [ t ] ( pg / ml)26.42
 3.141.03  3.0414.62  3.52**[t / c ] ratio1.6  0.221.12  0.150.45  0.22**ldh ( iu / l)4.71  0.5718.23  3.5513.51  3.56***all values represent as mean  sd.*p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 .
the levels of stress hormones were normal and within acceptable clinical 
 ranges for the conditions under which they were collected .
cortisol and free testosterone 
 concentrations were significantly increased ( p < 0.01 ) in the post - exercise recovery 
 samples compared to their pre - training values after four weeks of moderate intensity of 
 training , and there was a significant decrease in the t / c ratio ( p < 0.01 ) ( table 2 ) .
four weeks of moderate exercise training 
 also elicited significant increases in lactate dehydrogenase ( ldh ) concentration ( p 
 < 0.001 ) .
the correlation analysis revealed that the increase of cortisol was positively 
 ( p<0.001 ) related to the changes in total free testosterone , the t/ c ratio , and the 
 lactate dehydrogenase level in the post - training samples .
the pre - training samples showed no 
 relationship of cortisol with other stress biomarkers ( p > 0.05 ) ( table 3table 3.correlation of cortisol ratios with other stress biomarkers between pre- and 
 post - testvariablespre - training valuepost- training valuer(95%
ci)r(95% ci)cortisol vs. testosterone0.0175 ( 7398)0.25**94 ( 87100)cortisol vs. [ t / c ] ratio0.0680 ( 75100)0.53**89 ( 87100)cortisol vs. ldh0.0968 ( 56100)0.21**90 ( 86100)p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 .
all values represent as mean  sd.*p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 .
the endocrine system , by modulation of anabolic and catabolic processes , plays a major role 
 in the physiological adaptation to exercise training43 .
hormonal assays particularly anabolic ( testosterone ) and catabolic 
 ( cortisol ) hormones have been suggested as being valuable indicators of the exercise 
 intensity and work load .
the ratio between anabolic and catabolic hormones has been used to 
 determine the readiness status of individuals10 . whereas , the free testosterone to cortisol ratio is used as an 
 adaptation exercise index for males44 .
this study was performed to investigate the efficacy of 4 weeks of moderate aerobic 
 training on the levels of saliva stress hormones and lactate ( ldh ) activity as an early 
 detection of muscle injury among young adult men .
the results show that 4-weeks of moderate 
 aerobic training by young men led to significant changes in saliva stress hormones and 
 lactate level ( ldh ) .
it is an enzyme protein that 
 converts pyruvate to lactate , with concomitant conversion of nadh to nad45 . in this study
measurements of ldh following exercise have 
 been examined to seek evidence in support of the theory of muscle soreness .
distinguishable 
 elevation in aerobic enzyme capacity was observed following long or short - term aerobic 
 training in mammals46 , 47 .
previously , it was reported that in active muscles the 
 rate of lactate removal from the muscle cells into the blood increases following active 
 exercise intervention48 . therefore , the 
 increase of blood lactate is due to the release of lactate from muscles into the blood 
 following exercise .
exercise training produces changes in the concentration of several biologically active 
 molecules including cortisol and testosterone49,50,51 , 
 which play pivotal roles as catabolic and anabolic agents in gluconeogenesis via the 
 proteolytic pathway .
four weeks of moderate exercise training produced significant positive increases in the 
 concentrations of cortisol and free testosterone .
salivary testosterone increases found in 
 this study , coupled with the observed change in the resting cortisol level suggest an 
 enhanced anabolic environment .
these results were in agreement with previous studies that 
 reported that aerobic training increased the levels of cortisol and serum testosterone53 , 54 .
the increase noted in testosterone was probably induced by 
 adrenaline stimulation , the stimulatory effect of lactate , or the compatibility of 
 testosterone secretion55 .
changes in the levels of cortisol and testosterone depend on the intensity of exercise 
 training56 , 57 , as it was reported that exercise intensity of more than 60% 1rm 
 ( one repetition maximum ) increased the cortisol concentration significantly58 .
the testosterone - to - cortisol ratio ( t / c ) is considered to be a sign of the hemostatic 
 balance between both anabolic and catabolic states in athletes ; and this ratio plays a 
 significant role as a prognostic biological marker for insufficient recovery and 
 overtraining syndrome59 . in this study ,
significant decrease in the t / c ratio was found in young men following 
 4-weeks of exercise training , a result that was in agreement with other studies60 , 61 , and that the increase in cortisol following intensive endurance 
 exercise was accompanied with significant lowering of the t / c ratio as previously 
 reported62 , 63 .
however , the increase in the level of testosterone was 
 significantly related to the increase in the level of gonadotropin hormone after a 
 marathon64 . in this study ,
positive 
 relationships were found among cortisol , free testosterone , t / c , and ldh activity in young 
 men following 4-weeks of exercise training , as previously reported65 .
however , in the resting stage ( pre - exercise ) no 
 significant correlations were found among the studied stress hormones , similar to previous 
 reports33 , 34 . the positive relationship between cortisol and free testosterone
may be related to 
 stimulation of the adrenal gland in response to physical stress , since both hormones are 
 synthesized in the same cascade of reactions66 , indicating that stimulation of the adrenal gland via stressors 
 results in concurrent secretion of both hormones which ultimately increase in the blood67 .
also , the increase in free testosterone 
 may be related to changes in the binding affinity of the carrier protein as a result of 
 change in ph and temperature due to exercise .
this in turn would have decreased the level 
 and uptake of the carrier protein to hormones which would be finally liberated as free 
 hormones68 . in the same manner
, the positive correlation between post - exercise cortisol concentration 
 and ldh activity may be affected by several mechanisms including blood lactate 
 accumulation69 .
the findings of this 
 study also show that the increase of lactate at the end of exercise can elevate levels of 
 cortisol during recovery .
thus , the changes of salivary stress hormones along with ldh 
 detected in the present investigation may be related to the effect of short term aerobic 
 exercise . in conclusion ,
four weeks of moderate aerobic training three times per week , significantly 
 elevated the levels of stress hormones , cortisol , free testosterone , and ldh in young men , 
 with significant relationships among levels of cortisol , free testosterone , t / c , and ldh 
 activity .
however , more research is needed to validate these hormones as biological markers 
 of physical activity stress .
the 
 major limitation of this study is its low sample size , as only participants who were 
 measured at pre- and post intervention and follow - up were included .
also , this study 
 included only males which mean the results are gender specific .
furthermore , the exercise 
 duration was short , and the study should be repeated with longer exercise periods to measure 
 the effect of long term moderate aerobic exercise on stress hormones . finally , many studies 
 define exercise intensity by heart rate which requires measurement of oxygen consumption , 
 and this is an avenue of investigation we intend to pursue .
 [ purpose ] the purpose of this study was to evaluate the effect of 4 weeks moderate 
 aerobic exercise on outcome measures of saliva stress hormones and lactate levels in 
 healthy adult volunteers . 
 [ subjects and methods ] sixteen healthy students with an age 
 range of 1525 years participated in this study . 
 the participants performed an exercise 
 test of moderate intensity for 4 weeks , three times per week . 
 the exercise was treadmill 
 walking . 
 saliva concentrations of cortisol , testosterone and lactate dehydrogenase ( ldh ) 
 were measured before and after the 4 weeks of moderate aerobic training using immunoassay 
 techniques . [ results ] after 4 weeks of exercise 
 , there were significant increases in 
 cortisol , free testosterone levels , and ldh activity along with a significant decrease in 
 the ratios between testosterone and cortisol levels . 
 no significant correlations were 
 found among the studied parameters in the resting stage , a result which supports the 
 positive effect of exercise on stress hormones following 4 weeks of training . 
 [ conclusion ] 
 the results suggest that four weeks exercise of moderate intensity significantly affects 
 the salivary stress hormones of young healthy volunteers . 
 the data support the importance 
 of salivary stress hormones as potential biological markers especially for older ages . 
 
 however , more research is required to validate these biological markers which determine 
 the host response to physical activity . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION