the benefits of exercise may result from the direct effect on immune response modulations or from the mechanism of psychological effects of exercise.1 , 2 exercise may be considered a type of stress that affects immune response differently dependent on the duration , intensity and frequency of the stress . generally , many researchers found that low intensity and moderate intensity exercise improves immunity , conversely strenuous exercise and overtraining decrease immunity and raise infection risk.6 , 7 , 8 , 9 , 10 , 11 besides regular weight - bearing exercise , nutrition also plays an important role in influencing immune function status .
besides green tea and ginseng , honey is one of the nutraceuticals which are becoming more widely accepted as an adjunct to conventional therapies for enhancing general well being.12 , 13 honey contains mainly of carbohydrates that may elicit beneficial effects on reducing stress to the immune system .
this speculation is based on a study by nieman which mentioned that ingestion of fluids that contain carbohydrate can reduce perturbations in the immune system with fewer disturbances in blood immune cell counts , lower granulocyte and monocyte phagocytosis and oxidative burst activity , diminished pro- and anti - inflammatory cytokine responses .
honey also has properties of antioxidant,15 , 16 antimicrobial,12 , 17 , 18 , 19 anti - inflammatory and immunomodulatory which are believed able to enhance immune functions .
understanding the relationship between exercise , honey supplementation and immune response has potential implication for public health .
in addition to the important role of honey on the immune response , scientists also accept honey as a new effective medicine for many kinds of women related diseases .
therefore , health programs for women could be developed in view of vast advantages of honey on the immunes system .
previous study has been carried out to confirm the hypothesis that carbohydrates may influence exercise - induced immune changes.22 , 23 in addition , several studies revealed that honey is an effective carbohydrate source to be consumed at pre , during and post physical training and exercise in athletes.24 , 25 nevertheless , the combined effects of aerobic dance exercise with honey supplementation which is a source of carbohydrate on immune functions has not yet been investigated in non - athletes , i.e. sedentary women with age ranging from 25 to 40 year - old .
hence , the present study was proposed for determining the effectiveness of this combination on blood immune function parameters in adult women .
forty four physically healthy sedentary adult female participants , age between 25 to 40 years old from kelantan region , malaysia were recruited in the present study .
the inclusion criteria of the participants were : no health problems , non - smoker , not habitual consumer of honey daily , do not engage in any training program and do not exercise more than once per week .
physical activity readiness questionnaires ( par - q ) forms were answered by the participants to screen for any signs or symptoms of heart diseases , pulmonary or contraindications during physical activity for ensuring the participants were eligible to engage with the exercise training programme without risking their health condition , and limiting the medication drugs ' effect on the outcome later .
the qualified participants were matched in age , body mass , height and percent of body fat before they were randomly assigned into the experimental groups .
all participants were fully informed by the researcher about the nature of the experiments , purpose of the study , procedures , benefits , risks of feeling discomforts experienced in this present study before giving their written and signed formal consent .
the present study was approved by the human research ethics committee of universiti sains malaysia ( ethical approval number : usmkk / ppp / jepem[226.3(08 ) ] ) .
the human research ethics committee of universiti sains malaysia is in compliance with ich gcp guidelines . during the experimental period ,
the participants were provided checklists which have also been used in a published previous study , and they were required to record their participation , i.e. frequency of performing aerobic dance in a week and daily honey consumption rate in the checklists for ensuring their compliance and commitment to the present study .
please refer to the figure  1 for the flow chart of the experimental design for the study .
the participants were randomly divided into four groups with 11 participants per group ( n  =  11 ) : 8 weeks of sedentary without supplementation control ( con ) , 8 weeks of honey supplementation ( h ) , 8 weeks of aerobic dance exercise ( d ) , and 8 weeks of combined aerobic dance exercise and honey supplementation ( hd ) groups .
participants in the control group ( con ) did not perform exercises nor take any honey supplementation .
meanwhile , participants in the honey group ( h ) consumed honey drink for 7 days per week for a total of 8 weeks .
participants of aerobic dance exercise group ( d ) performed one hour aerobic dance exercise per session , 3 times per week for 8 weeks .
participants in combined aerobic dance exercise with honey supplementation group ( hd ) performed aerobic dance exercises one hour per session , 3 times per week for 8 weeks and consumed honey drink 7 days / week for 8 week with dosage similar to honey group ( h ) .
the participants in hd group were required to consume honey drink 30  min before performing aerobic dance exercise on the exercise days .
the power of the study was set at 80% with 95% confident interval and 30% of effect size .
thus , the actual numbers of participants recruited were 11 participants per group , and the total numbers of participants recruited in this study were 44 participants .
the participants of aerobic dance exercise group ( d ) and combined aerobic dance exercise with honey supplementation group ( hd ) were required to have aerobic dance sessions for 3 sessions per week , one hour per session ( from 5.30  pm to 6.30  pm ) for 8 weeks .
the aerobic dance exercise program of this study consisted of 2 sessions of  high and low impact and one session of a 
the 1-hour session started with 1015  min of warm up period , 3035  min of aerobic dance activities , and ended with 57  min of cool down .
the activities prescribed in the present aerobic dance exercise program involved continuous , controlled movement of legs and trunk , and intermittent movement of arms .
movements involved were side stepping , fast walking , forward and backward stepping , leg lifts , placing foot to the front , side and behind , knee bends , forward and side - lunging , heel raise and also high impact movement such as jumping . in the high - impact and low - impact aerobic dance exercise sessions
, participants were required to do upper and lower limb movements according to the beats of the music played , which ranged from slow to fast . in the  step board exercise sessions , participants were required to step up and down the step board while dancing .
the intensity of aerobic dance exercise was estimated by using heart rate monitor ( polar , s710 , usa ) worn by participants throughout the dancing sessions .
besides , the participants were given pre - recorded cd containing aerobic dance workout , and they were required to follow the workout in the cd given at home if they missed any of the aerobic dance sessions .
a honey drink was consumed by the participants in the honey ( h ) group and combined aerobic dance exercise and honey supplementation ( hd ) group in a dosage of 20  g27 , 28 of malaysian local gelam honey diluted in 300  ml of plain water , for 7 days per week for a total of 8 weeks . on the exercising days , the participants of the combined aerobic dance exercise and honey supplementation ( hd ) group were required to consume honey drink 30  min before performing aerobic dance exercise . before the 8 weeks of experimental period , participants ' anthropometric measurements such as body height
the participant 's body heights were measured by using a stadiometer ( seca 220 , germany ) , the body weight and percentage body fat were measured by using a digital body composition measuring device ( tanita tbf-410 , japan ) . before and after the 8-week of experimental periods , participants were seated and a 2  ml of venous blood sample was taken from an antecubital vein after 8-hour overnight fast ( drinking plain water was allowed ) .
the blood was withdrawn by the laboratory technologists in the sports science laboratory , universiti sains malaysia .
blood taking sessions for participants in d and hd in post test were carried out were carried out at 8.30 the next morning after performing aerobic dance exercise , i.e. 14  h post exercise .
blood samples were used to determine the levels of full blood counts such as white blood cells , neutrophils and total lymphocyte counts by using an automated haematology analyser ( sysmex xs-800i ) . meanwhile
, the concentration of blood immunophenotyping parameters , i.e. total t lymphocyte ( cd3 ) , t helper ( cd4 ) , t cytotoxic / suppressor ( cd8 ) and natural killer ( nk ) cell counts were analysed by using a flow cytometer ( bd facs cantor ii , becton dickinson , usa ) , and three - colour direct immunofluorescence reagent kit ( bd tritest , usa ) .
statistical analysis was done by using statistical package for social science ( spss ) version 18.0 .
two way repeated measure analysis of variance ( anova ) was performed to determine the significance of the differences between and within groups .
the difference was considered statistically significant at p  <  0.05 . confounding variables such as participants ' age , body mass , height and body fat
the participants were matched in age , body mass , body height and body fat before they were randomly assigned into the experimental groups .
one - way analysis of variance ( anova ) was performed to ensure that there were no significant differences in the aforementioned confounding variables among the groups at the beginning of the study .
a total of forty healthy sedentary adult women ( mean age 29.7    5.3 years ) completed the present study .
two participants from honey supplementation group ( h ) and two participants from combined aerobic dance exercise with honey supplementation group ( hd ) were unable to continue the program due to pregnancy and personal reason during the experimental period .
participants ' mean body height in con , h , d and hd was 154.2    5.6  cm , 153.8    4.8  cm , 154.6    6.1  cm , and 156.4    6.0  cm respectively .
the mean body weight and percentage of body fat of the participants were 56.0    9.9  kg and 32.5    9.8 % in con group , 54.5    7.8  kg and 33.0    7.2 % in h group , 55.3    5.0  kg and 32.7    5.0 % in d group , and 53.4    7.7  kg and 30.0    7.4% in hd group respectively .
there were no significant differences ( p  >  0.05 ) between groups in means age , body height , body weight and percentage body fat at the beginning of the experimental period .
mean concentration of white blood cells ( wbc ) , neutrophils and lymphocytes counts of all groups are presented in table  1 . at pre test ,
wbc count concentrations were significantly higher ( p  <  0.05 ) in hd , d and h groups compared to con group respectively , and this measured parameter was also significantly higher ( p  <  0.05 ) in d group compared to h group . at post test , mean wbc count concentration was significantly higher ( p  <  0.05 ) in hd group compared to con group .
there were no statistically significant main effect of time ( p  >  0.05 ) on wbc count between pre- and post tests in all the groups except for d group , in which wbc concentration was significantly lower ( p  <  0.05 ) in d group after 8 weeks of intervention in post test compared to pre test value .
neutrophil counts were significantly higher ( p  <  0.05 ) in d group compared to con group at pre test . however , there were no significant simple effect of intervention ( p  >  0.05 ) on neutrophil counts between all the experimental groups at post test .
there was statistically significant main effect of time ( p  <  0.05 ) on neutrophils counts between pre- and post tests in d group , in which d group exhibited statistically significant lower ( p  <  0.05 ) neutrophil counts after 8 weeks compared to pre test value . in lymphocyte counts ,
there were significantly higher ( p  <  0.05 ) values in hd , d and h groups compared to con group respectively at pre test . at post test , there were significant difference ( p  <  0.05 ) between hd group compared to con group . however , there was no statistically significant main effect of time ( p  >  0.05 ) on lymphocyte counts in all the groups .
the percentage increase in lymphocyte counts after 8 weeks of study was the highest ( + 11.07% ) in hd group compared to the other three groups .
mean concentration of t lymphoctyes and natural killer ( nk ) cells of all groups are presented in table  2 . regarding total t cells ( cd3 ) counts
, repeated measures anova revealed that at pre test , total t cells ( cd3 ) count were significantly higher ( p  <  0.05 ) in h group compared to con group .
however , there were no significant simple effect of intervention ( p  >  0.05 ) on this cells count between all experimental groups after 8 weeks of experimental period at post test .
additionally , there was no significant main effect of time ( p  >  0.05 ) on post test mean total t cells ( cd3 ) counts compared to pre - test mean for each experimental group . at pre test ,
t helper / inducer ( cd4 ) count were significantly higher ( p  <  0.05 ) in h group compared to con group ( table  2 ) .
however , there were no significant simple effect of intervention on this cells count ( p  >  0.05 ) between all experimental groups after 8 weeks of experimental period in post test .
there was no significant main effect of time ( p  >  0.05 ) on post test mean t helper / inducer ( cd4 ) counts compared to pre - test mean for each experimental group .
it was found that hd group showed highest percentage increases among the groups in cd4 with  + 16.20% .
mean t cytotoxic / suppressor ( cd8 ) count of all groups are presented in figure  2 . at pre test ,
t cytotoxic / suppressor ( cd8 ) count were significantly higher ( p  <  0.05 ) in hd and h groups compared to con group respectively , and this measured parameter was significant lower ( p  <  0.05 ) in d group compared to h group . at post test ,
t cytotoxic / suppressor ( cd8 ) count were significantly higher ( p  <  0.05 ) in hd group compared to d and con groups respectively , and this measured parameter was significant higher ( p  <  0.05 ) in h group compared to con group .
a statistically significant main effect of time ( p  <  0.05 ) on t cytotoxic / suppressor ( cd8 ) count was found between pre- and post tests in hd group , where the cd8 count in hd group was significantly higher ( p  <  0.05 ) in post test than pre test .
it was also found that the percentage increase in t cytotoxic / suppressor ( cd8 ) counts after 8 weeks of study was the highest ( + 19.98% ) compared to other experimental groups .
mean natural killer ( nk ) cells counts of all groups are presented in table  2 .
the results showed that natural killer ( nk ) cells count were significantly ( p  <  0.05 ) higher in d group compared to con group before and after 8 weeks of experimental period .
there were no statistically significant main effect of time on natural killer ( nk ) cells counts ( p  >  0.05 ) between pre- and post tests in all the groups .
generally , physical activity is a type of stress and able to reduce wbc and neutrophil counts.6 , 7 in the present study , it was found that there was no statistically significant change in wbc and neutrophil counts in hd group . however , wbc counts decreased significantly by  14.83% and neutrophil counts also decreased by  25.86% in exercise alone ( d ) group in post - test compared to pre - test .
the reduction in wbc with exercise observed in the present study is inconsistent with most of the published studies which reported increases in total leukocytes and leukocytes subsets after exercise,31 , 32 , 33 and the present findings may reflect that both the duration and the intensity of exercise in the present study need to be considered .
the questions raised were whether the participants in the exercise alone group perceived the prescribed exercise in the current study as a type of stress , even though the participants ' average heart rates recorded during exercise were ranging from 120 to 140 beats  min , which was equivalent to 60%70% of the participants ' maximum heart rate , reflecting that the intensity of the prescribed exercise in the present study was considered moderate . the reductions in wbc and neutrophil cell counts in exercise group may imply that the prescribed exercise of this study may have acted as a stress to reduce the wbc and neutrophil counts .
this speculation is based on moynihan et  al . , which mentioned that the circulating numbers and functional capacities of white blood cells may be decreased by repeated bouts of intense , prolonged exercise .
the reason is probably related to increased levels of stress hormones during exercise and entry into the circulation of less mature white blood cells from the bone marrow . additionally , neutrophil is reported to be intensity dependent generally in which exercise at low to moderate intensity could enhance some aspects of neutrophils function , while maximum exercise suppressed neutrophil functions,6 , 7 and can cause functional alterations in neutrophil function .
it was hypothesized that when exercise and honey supplementation combined together in the present study , this combination may enhance immune function of the participants greater than if exercise or honey supplementation is carried out alone .
the notable finding of the present study was that there were statistically significant increases in t cytotoxic ( cd8 ) counts in hd group after 8 weeks of experimental period , implying that honey supplementation combined with aerobic dance exercise increased cd8 t cell counts greater than consumed honey and performed aerobic dance exercise alone .
similarly kwon et  al . , reported that four weeks soybean supplementation combined with moderate intensity endurance swimming exercise could prevent impairment of t cell through changes of cd4/cd8 ratio in rats .
therefore , it is suggested that honey and its constituent also have the beneficial effects on cd8 t cell counts .
the precise mechanism for inducing the beneficial effects on this immune function parameter in the present study is unclear .
however , it is speculated that during exercise , more blood is needed by participants ' body , therefore more vital nutrients in honey have been absorbed into participants ' body during exercise .
these vital nutrients enhanced immune functions of the participants by increasing the lymphocyte cell counts .
another notable finding of the present study was that the percentage increases in total lymphocyte , t helper ( cd4 ) , and t cytotoxic ( cd8 ) cell counts after 8 weeks were the highest in hd group among all the groups , even though statistically significant increases were not observed .
collectively , these findings may indicate that combined honey supplementation and aerobic dance exercise may have potential to enhance immune functions compared to honey supplementation alone or exercise alone .
it is speculated that carbohydrate contained in honey may be able to modulate immune function either by reducing the stress effect of exercise , nehlsen - cannarella et  al .
also reported that carbohydrate ingestion is associated with higher plasma glucose concentrations and it could attenuate stress hormones . in our present study ,
the findings reflect the effects of two months honey supplementation and aerobic dance exercise on the measured parameters , but not the acute effects such as a single session of honey supplementation and aerobic dance exercise on immune function parameters , and this may cause discrepancies between the present study results and other previous studies . for instance , natale et  al . , found that exercise induced a significant increase in total t lymphocyte ( cd3 ) cell counts immediately after exercise .
nevertheless , 3  h after exercise , the cd3 cell counts for peak aerobic exercise are 35% below baseline
. they also found that circulating cd4 ( t helper ) cell counts showed a significant increase with peak aerobic exercise and prolonged exercise but not for resistance exercise , and the counts were subnormal 3  h after the peak aerobic exercise . additionally , significant increased in cd8 t cell counts were observed immediately after exercise , but were restored to baseline 3  h after exercise .
their study have shown the changes of immune parameters during and a few hours after exercise , where the present study found that the changes in immune parameters occurred 14  h after the last exercise session of 8 weeks ' intervention .
additionally , it was suggested that the difference in sampling time is one of the possible reason for variation in blood analysis results . in the present study ,
it is believed that the sampling time used in the present study may have caused absence of some significant changes in certain measured immune parameters .
mobilisation of nk cells is one of the most robust changes to the immune system in response to physical exercise.39 , 40 the striking exercise - induced increase of nk cells count in peripheral pool is thought to translate into enhanced immune function , while post - exercise reduction in blood nk cells is proposed to increase one 's susceptibility to infection .
one of the most notable findings in the present study was that there was no significant changes in nk cells count in hd group , however great increase ( + 0.76% ) in nk cell counts could be observed in d group with greatest percent difference between pre and post test among the groups .
based on this finding , aerobic dance exercise alone may have potential to increase nk cell counts .
increase in nk cells count resulted from exercise has been reported by shephard and shek , and timmons .
elevated nk cells in response to acute exercise were found in adults and children . in another study ,
peak aerobic exercise induced significant increase in circulating nk cell counts immediately after exercise , however nk cells counts returned to baseline by 3  h post exercise .
these changes are probably due to an enhanced state of readiness against potentially harmful pathogen in human .
on the other hand , it was reported that high intensity exercise could induce suppression of nk cell function .
the absence of reduction in nk cells induced by the aerobic dance exercise prescribed in the exercise group of the present study may reflect that the exercise intensity perceived by participants in the exercise group was not high enough for reducing the nk cells . in the present study
, it was found that nk cells decreased over time in hd group . a previous study which reported that combined carbohydrate intake with 60  min and high - intensity cycling was not associated with an alteration in innate immunity .
nevertheless , previous study mentioned that the effect of exercise on nk cell activity can not be denied .
furthermore , timmons and bar - or showed that nk cell function is neither enhanced nor reduced following exercise in young girls , however carbohydrate intake attenuates the nk cell subsets responses to exercise in children . in the present study ,
nk cells count reduced when exercise combined with honey ( hd ) . additionally , reduced in nk cells
these observations were in agreement with statement mentioned by nieman that by maintaining higher plasma glucose levels and thus attenuating the cortisol and growth responses during exercise , carbohydrate beverage ingestion may reduce stress to the immune system in individuals . in general
, the discrepancy of the measured parameters between the present study and previous studies may be due to differences in type of exercise , duration of exercise , age range , types of nutritional supplementations given and particularly time of blood withdrawal after exercise .
one of the limitations of the study is that despite statistical analysis of one - way anova was performed to minimize the differences in the measured parameters among the groups at the beginning of the study , differences in certain measured blood parameters between the groups during pre test could not be avoided .
thus , the present study focused on the comparison of the variables between pre- and post - tests in each group .
it is suggested that future studies with multicentric experimental conditions such as different types of exercise and nutritional supplementation , prolonged intervention period and repeated blood withdrawing after exercise , as well as larger sample size are needed for details and comprehensive findings .
the present study found that combination of aerobic dance exercise and honey supplementation elicited more beneficial effects on immune functions generally compared to aerobic dance exercise or honey supplementation alone in sedentary women .
therefore , supplementation of honey drink with 20  g of honey diluted in 300  ml of plain water combined with 3 days per week of aerobic dance exercise has potential to be proposed for formulating guidelines in planning exercise and nutrition promotion program for increasing immune functions in sedentary women .
 to date , information on the effectiveness of combined aerobic dance exercise with honey supplementation on immune function in women is lacking . 
 the present study investigated the effects of 8 weeks of combined aerobic dance exercise and honey supplementation on blood immune function parameters in adult women . in this study , forty four healthy sedentary women ( 2540 year - old ) were assigned into four groups with n  =  11 per group : sedentary without supplementation control ( con ) , honey supplementation ( h ) , aerobic dance exercise ( d ) and combined aerobic dance exercise with honey supplementation ( hd ) groups . 
 aerobic dance exercise was carried out for one hour per session , three sessions per week for eight weeks . 
 honey drink was consumed by h and hd groups , in a dosage of 20  g of honey diluted in 300  ml of plain water , consumed 7 days a week for 8 weeks . in hd group , 
 the participants were required to consume honey drink 30  min before performing exercise . before and after 8 weeks of experimental period 
 , blood samples were taken to determine the concentrations of immune parameters which include full blood counts and immunophenotyping measurements . 
 it was found that after 8 weeks of experimental period , there were statistically significant increases in t cytotoxic ( cd8 ) ( p  <  0.05 ) in hd group . 
 additionally , the percentages increase in total lymphocyte counts , t helper ( cd4 ) , and t cytotoxic ( cd8 ) counts after 8 weeks were the highest in hd group among all the groups . as conclusion , 
 combined aerobic dance and honey supplementation may have potential to enhance immune functions in women . 
Introduction
Methods
Results
Discussion
Conclusion
Competing interests