Effects of Acute Supplementation of Honey on Endurance Performance 27 Original Article Effects of Acute Supplementation of Honey on Endurance Performance in Male University Students

Full text

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
27
Original Article
Effects of Acute Supplementation of Honey on Endurance
Performance in Male University Students
Ishita Bhattacharjee and Amit Bandyopadhyay*
Sports and Exercise Physiology Laboratory,
Department of Physio logy, Univers ity of Calcutta,
Univer sity Colleges of Science and T echnology,
92 A.P.C. Road, Kolk ata – 700009, India.
Abstract
Backgroun d: Ingestion of combination of carbohydrates (CHO) is beneficial f or exercise performance. Honey
is a natural readily available source of C HO mixtur e which possesses anti-oxidant and imm une boosting
properties, so it might e nhance endurance per formance by ma intaining bloo d glucose level during ex ercis e
and reducing exercise induced oxida tive s tress. The present s tudy was conducted to investigate the efficacy
of acute suppleme ntation of honey on endurance perform anc e in healthy male individuals.
Metho dolog y: In this PL-contr olled d ouble-blind randomize d study, 12 healthy males (20-25 year s) were
supp lemented with Honey 30 g or Honey 60 g before they per for med the treadm ill running at a speed that
elicited 80% of HRmax after an overnight fast. Pre–exercise HR, body temperat ure and RPE were also
reco rded. Blood sa mples were drawn bef ore supplem entation and at the tim e of exhaustion for analysis of
glucose, insulin, lactic acid, free fatty acid, superoxide dismutase, lipid peroxidation, catalase and total
thiol. Data were analyzed by two way RM ANOVA followed by post hoc test.
Results: Endura nce tim e showed significant (p<0.05) diff erence be tween the PL and honey trials as well as
between H30 and H60 trials. Changes in working heart rate (HR) were significantly (p<0.05) diff erent over
time in the sam e experim ental trial com pared to the respective resting value. W orking HR had no significant
diff erenc e between H30 and H60 trial s, except at 9 0th m in and at the tim e of exhaustion. H30 trial showed
significant (p<0.05) difference in working HR in comparison to PL trial at 0th, 20t h, 40th , 50th , 60th and 80th min.
Working HR of H60 trial were also sign ificantly (p<0.05) dif ferent in compariso n to PL trial. RPE was
significantly (p<0.05) different at the time of exhaustion in comparison to the 10th min value of the corresponding
trial. Values of lactate, glu cose, insulin, free fatty acid, lipid peroxidase, total thiol, catalase and superoxide
dismutase depicted significant differenc e not only between H30 and H60 trials but also between PL trial and
two differ ent honey trials.
Interpretation and con cl usio n: Acute honey supplementation has significant beneficial effect on endurance
perf orm ance and biochemic al blood par ameters. So it can be used as a natural ergogenic aid to boost
exercise performance.
Indian J Physiol Pharmacol 202 0; 64(1) : 27–37
*C o r r espon d i n g au thor :
Dr. A mit Bandyopad hy ay , S ports a nd Exercis e P hy siolo gy La borat or y, De partm ent of Physiol og y, Uni ve rs ity o f C al cut ta ,
University Colleges of Sci en ce an d T echnol ogy, 92 A.P.C. Road, Kolkata – 700009, India. Ema il : a bp hys@ ca lu ni v.ac.in
(Received on O ct . 1, 2019)

2 8 Bhattacharjee and Bandyopadhyay Indian J Phys io l Ph arma col 20 20; 64(1)
of fr ee r a d ic a ls a n d th u s im pr ove e x er c is e
perform ance an d recovery time.
Chronic supplementation of honey enhanced exercise
perf orm ance by improv ing power output, hear t rate
and blood glucos e level during en durance trials (1,
6). However, the eff ect of acute supplementatio n of
honey on enduranc e performance has no t yet been
explored. Moreover, literature regarding the effic acy
of acu t e supplem e n t at i o n of h on e y on In d i a n
population is also unavailable. Therefore, the present
stud y was aim ed to investigat e th e ef fects of acute
supp lementation of honey on endurance capacity in
young male University stu dents of Kolkata, India.
Materials and Methods
Sel e cti o n o f the s u b jec t
Sa mpl e Siz e C a lcu lat ion :
Twelve [12] m ale recreationa l athletes , who jog at
le ast 3 0 minutes t wice in a we ek ac c ordi n g to
Jack son (8), belonging to the age group of 20 to 25
years were recruited in the study f rom t he post-
graduate students’ population of University of
Calcutta, Kolk ata, West Bengal, India. Individ uals
who were undergoing any medication or suffering from
any healt h h a z a r d l i ke hyperte n s i on , a s th m a,
di a bete s , bro nchit i s, an emia, c a rdiac problems,
kidney or liver diseases or any other m ajor diseas es
were excluded to tak e part in the study.
The sam ple size was calculated by using PS Power
and Sample Size Calculation version 3.0 ( 9) where
the confidence interval was set at 95%. The calculated
number of subjec ts was nine [9]. T his study was
exec uted with 12 subjec ts (n=12).
The entire experimental procedure along with probable
menace aspect was clea rly clarified to each subject
Introduction
Consu m ptio n o f carbo hydrat e before a nd during
endurance exercise impr oves ex ercise performance
(1, 2). Honey is a natur al yellowish or brown visc ous
fluid produced by honey bees, Apis mellifera, from
the nectar of f lowers. This supersaturated solution is
a mixt ure of c arbo hydrates m a inly f ruct o se a nd
glucose along with various c ompounds like proteins,
or g an i c ac ids , vi t a m i n s , m in er a l s, e nzym es ,
polyphenols, etc. (3). Honey as a nat ural sweeten er
provides 64 calories of energy per tablespoon which
is generally absorbed within 15 mins and is released
into the blood stre am at a steady rate throug hout
the exerc ise tenure if ingested prior to perform ance
and may helps to maintain blood glucose level during
endurance perf orm ance a nd glycogen restoration
foll o wing e x e rc ise (3 ) . It has m uc h thera p e u t ic
signif i c a n c e w h i c h i n c l u d e s a n t i- a l l e rg i c , a n t i -
inflam matory, anti-microbial, anti-parasitic, anti-viral
properties, probiotic effect, eff ect s on eye and oral
health, wound healing, anti-cancer, anti-tumor, anti-
mutagenic, diabetes and weight management, reduce
coronary heart dis ease a nd m aintains general health
and nutrition (3). Several researc hes revealed that
honey possesses a ntioxid ant property due to the
pres ence of flavonoids and polyphenols which help
in the scavenging of superoxide anions (4). Generally
the dark er the hone y, the higher is its phenolic
con t e n t and its a n tioxi d a t i ve p ow e r (5) . T his
antioxidant property influences the activity of glucose
oxidase, catalase, ascorbic acid, flavonoids, phenolic
acids, carotenoid derivatives and organic acids (5,
6). The hypopharyngeal glands of honeybee workers
produce glucose oxidase and deposit it into honey
where this enzyme acts as a nat ural preservative.
Gluc ose oxidase remains present at the sur fac e of
th e ho ney and conv erts atmosp heric oxygen to
hydr oge n p eroxide that ac ts as an a nti micr obial
barr ier (7). So, it is quite justif ied to speculate that
honey may neutralize the exercise induced generation
Abbreviatio ns us ed: CHO- carb ohydra te, PL- placebo, H30- honey 30g, H60- Honey 60 g, HR-Heart R ate,
HRmax - heart rate maximum , RPE- rate of perceived ex ertion, LA- lactic acid, FFA-free fatty acids, LPO- lipid
peroxidation, SOD - superoxide dismutase, RM- Repeated m easures, ANOVA - analys is of variance.

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
29
pri o r to signi n g the co n s e n t form. The eth i c a l
clearance for the research work was approved by
the H u m an Eth i c s C om m i t t e e , Depa r t m ent o f
Physiology, Universit y of Calcutt a.
Dos e s :
Honey was supplemented at two different doses, i.e.,
30 g (H30) and 60 g (H60) (3). Artif icial sweetener
(which does not contain any calorie substance) was
used as placebo (PL) to match the taste with the
honey. Each of the supplements was diluted in 250 ml
plain water and it was supplied in opaque bottles to
omit the colour differences. Since raw honey causes
allergic reactions in some cases, marketed honey
by Dabur India Ltd. was u sed in the present study.
Each 100 g of Dabur honey was composed of 80 g
natural s ugars , 17 m g sodium , 138 mg potassium ,
13 m g calcium, 1.5 mg iron and 5 mg phospho rus.
This yields 320 kcal of energy ( 10).
Ex p eri m e nta l Des i gn:
The subjects were ass ess ed in a random ized, PL–
cont rolled d ouble blind m anner. Each subject cam e
to the laboratory for 6 times. They had to per form
treadmill runnin g on a motorised treadmil l ( Viasys,
Germ any). The first three vis its (Visit 1, 2 an d 3)
were pre–e xperimen tal trials in which the subjec ts’
fitn ess were measured and they were fam iliaris ed
with the experimental protocol and the speed of the
treadmill was determ ined that elicited the subjec t’s
80% of HRmax. Three experim ental trials with different
supplements (PL / H30 / H60) were conducted in the
next thr ee visits (Visit 4, 5 and 6) with at least 4
da y s g ap b e t we e n t he d a ys o f c on s ec u ti v e
exp e r i m enta l t r i a ls . A t l e a st 7 d ay s g a p w a s
main tained between pre -trial and experime ntal trial
protocols.
Subjects reported to the laboratory at 8:00 am on all
occa sions after an overnig ht fast of 12 hours. The
entire study was conducted dur ing March 2017 to
August 2017 in a laboratory tem perature of 29-32ºC
and relative humidity of 58-78%.
Pre p a rat i o n o f s u b ject s :
Subjects were ask ed to refrain f rom he avy exe rcise
for 24 hours before the days of the trials. Their food
diary and physica l activit y diary (11 ) for the last 72
hours was collected. Subjects wer e requested to
reco rd their food intak e for the last 3 days prior to
the first trial and repeat the sam e d iet over 3 days
befo re the days of the consec utive trials (11). The
mai n pu rpose wa s to dim i nis h the in equality in
muscle gl ycog en level of th e su b j ect’s prio r to
exerc ise. They were reminded to ensure compliance.
The age of the su bjects was calculated from the
date of birth as r ecorded in their identity card issued
by the Univer sity a uthority. The body height was
meas ured with the subject standing barefoot with an
accuracy of ±0.50 cm, an d t he body m ass was
measured to an acc uracy of ±0.1 k g by using a
weight measu ring instrument f itt ed with a heig ht
measuring r od (Avery India Ltd., India) with th e
subj ect wearing m inimum c lothing.
Pr e -tr i a l p r o toc o l :
The main objective of the pre-trial protoco l was to
familiarise the su bject with the exer cise protocol as
well as to select the speed of the treadmill at which
the subject attained his 80% of HRmax or age-predicted
maxim um heart rate (220 – Age). First the subject
un d e rwen t a ph y s i cal e x am i n atio n pr o c e dur e
(measurement of heart rate, blood pressure, recording
of ECG, m easurement of body height, body weig ht
and s kin tem perat ure) to ensure that th e subject
was fit to participate in the investig ation. Af ter that
the Polar heart rate monitor was placed on the chest
surf ace of the subject to record the heart rate. The
warm up exercise comprised of treadm ill running at
a speed 5 km.h-1 at 0% elevation for 5 m ins. During
the exercise trial the heart rate of the subject was
monitored at every 10 min. T he speed of the treadmill
at which the subject attained a steady state heart
rate of 80% of his HRmax was noted.
Tr ial Pro toc ol :
Subjects were tested in the morning at 8 am onwards
afte r 1 0 - 1 2 h o u rs o f o ve r n ig h t f as t . B u t t h ey
were perm itted to drink plain water (500 m l) and
were given a piece of bread on arrival in the laboratory
(11).

3 0 Bhattacharjee and Bandyopadhyay Indian J Phys io l Ph arma col 20 20; 64(1)
Fig. 1 : The e xperim en tal de si gn of th e st udy.
[PL-Pl ac eb o, H30-Ho ne y 30 g, H60-Honey 60 g]
Du r i n g t h e expe r i m e nt:
Just before the com mencem ent of e xercise (0 min),
8 ml of venous blood sample was withdrawn from
the antec ubital vein by a compe tent phleb otomist
us i ng s t and a rd i z e d ve n ipu n ctu r e te c hni q ues .
Supplem entation was given in the form of solution at
this time in an opaque bottle, one hour bef ore the
exper i m en t a l t r ia l . A f te r w a r m up the su b j e c t
perf orm ed treadmill runnin g at the specifie d speed
that elici ted 80% of ag e predicted heart r ate in the
pre- exerc ise trial. T he exerci se continued till onset
of fatigue that was ensur ed by subjects’ own volition
in spite of verbal encouragement along with attainment
of peak heart rate that was within a range of HRmax±10
beats. T he blo od s ample was collected at the point
of exhaustion. During th e t ria ls, reading of f luid
sensory scale (to measure gastrointestinal tolerance)
was collected at an int erval of 20 m ins. Heart rate,
skin temperature, room temperatur e, room relative
humidity and RPE (Borg’s Scale) were colle cted at
intervals of 10 mi n. Cool water (4-8°C) @ 3 ml per

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
31
Fig. 2 : Experi me nt al tr ial Pr ot oc ol.
[Here PL-Pl ac ebo, H3 0-Ho ne y 30 g, H60-Honey 60 g ]
kg of body weight was given to the subjec t at an
interval of 20 minutes to stay away from any possible
undesirable effects of dehydration (11). Once the
subj ect s topped running, he was req uested to cool
down for about 2-3 minut es on the treadmill at a
walkin g speed of 4-6 km.h–1 .
Af ter th e E x p erime nt:
Body weight was taken afte r the test. Sweat rate
was determined by difference in nude body weight
and the am ount of fluid cons umed (12) . Bes ides, 8
ml of venous blood was drawn at the time of fatigue.
450 m l of water for every 0.45 kg loss of body weight
lost was given to the subject to prevent d ehydration
(13). Same procedure was repeated with the s ubject
in t he subs equent trial(s ).
Bl o o d A nal ysi s M eth o d :
8 m l of ven ous blood was drawn during each blood
sam pling of which 1 m l was taken in EDTA vacutainer
for plasm a lactate (LA ) analysis and 7 ml was taken
in normal vial without EDTA for serum pr eparation.

3 2 Bhattacharjee and Bandyopadhyay Indian J Phys io l Ph arma col 20 20; 64(1)
Collec ted blood was allowed to stay untouched for
at l east 2 hours. After 2 hours, supernatant serum
was collected and centrifuged (Remi centrifuge, India)
at 3000 r pm for 10 min . The s u p ernat a n t wa s
collec t e d a nd t r an s ferred t o p r o pe r l y lab e l l e d
eppendorfs for biochemical analysis. Serum was used
for the analysis of free fatty acid (FFA), glucose,
insulin, superoxide dismuta se (SOD), catalase, lipid
peroxidation (LPO) and total thiol by enzym e assay.
Oxid ative stress was assessed by LPO produc t in
the for m of thiobarbituric acid reactive substa nces
(TBARS), enzymatic anti-oxidants in the form of SOD,
cata lase, and total thiol.
Es tim a ti o n o f T o ta l Pro tei n:
Total serum protein concentration was determined
by the meth od of Lowry e t al. (14).
Es tima tio n o f Gl uco s e:
Plasma glucose was estim ated by UV test- Ezymatic
reference method with hexokinase (15, 16) on Roche/
Hitach i cobas c 31 1 systems. Precis ely, hexokinase
catalyzes the phosphorylation of glucose to glucose-
6- p ho s ph a te by AT P . Gl u c os e- 6 -p h o sp h at e
dehydrogenase ox idizes glucose- 6-phosphate in the
pres ence of NADP to gluconate-6- phosphate. No
other carbohydrate is oxidized. The rate of NADPH
formation during the reaction was directly proportional
to the glucose concentration and it was measur ed
photometrically at 340 nm . The values are expressed
in Units mmol/L.
Es tima ti o n o f I ns u lin :
Serum insulin was m easured according to Sapin et
al. (17) by Electrochemiluminescence immunoassay
(ECLIA) by using Roche Elecsys Insulin 2010 reagent
kit (Roche Diagnostics Corporation, Indianapolis, IN
46250). The Elecs ys Ins ulin assay e m ployed two
monoclonal ant ibodies which together were specific
for human insulin.
Es tim ati on o f Pla sma F re e Fa tty Aci d :
FFA present in serum was estim ated by the method
of Kwon an d Rhee (18). An aqu eous solution of
cupr ic acetate was prepared [5% (w/v ), pH 6.1 by
using pyridine] and filtered. The so lution did not
require any other color reagent. The standard curves
of FFAs versus absorbanc e were determined b y
measuring the absorbance of isooctane s olution at
715 nm against the c ontro l which contains no free
fatty acids. The values are expressed in Units m mol/
L.
Est i m ati on of S upe r o xide d i s m u ta s e :
SOD was a ssessed ac cording to the m ethod of
Mark lund and Marklund (19). 1 ml Tris-HCL-EDTA
buff er (50 mM, pH-8.2) an d 17 µl serum was mixed
well i n a m i c r o- c e n t ri f u ge t u b e ( wr a p p e d with
alum inum foil) a nd inc ubated in dark at 25ºC for 10
min. Pyrogallol wa s prepared fr eshly and kept in
dark due to its light sensitivity. Th e enzyme activity
was meas ured at 420 nm and was e xpres sed as
units/mg of protein. One unit of enzyme is defined
as the activity that inhibits autooxidation of pyrogallol
by 50%.
Es tima tio n o f Cat ala s e:
The catalase activity was measured by the method
of Claibor ne (20). The assay mixture consisted of
1.95 ml phosphate buf fer (0.05 mM, pH-7.0), 100 µl
serum and 1.0 ml H2O2 (0.019 M). The disappearance
of H2O2 was monitored at 240 nm wavelength at an
interval 1 min for 5 m ins. Catala se activity was
calc ulated in term s of nmol H2O2 consumed/m inute/
mg protein.
Es tim a ti o n o f L i pid Per o xid ati on:
LPO was estimated by the method of Wright et al.
(21) . Reactio n mixture containi ng 580 µl phosphate
buffer ( 0.1 M, pH-7.4 ), 200 µl FeCl3 (100 mM), 300 µl
ser um and 200 µl ascorbic aci d (1 0 0 mM) was
incubated at 37ºC in a water bath for 1 hour. The
reaction was stop ped b y t h e ad ditio n of 1.0 m l
trichloroacetic a cid (TCA) (10% w/v), followed by the
addition of 1.0 ml thiobar bituric acid (0.67% w/v).
The test tube was placed in a boilin g water bath for
20 m in and coole d and t hen centrifuged at 3000 × g
for 10 m in. Th e s upern atant containin g TBARS was
assessed by measuring the optical density at 535 nm.
The result was expres sed as nmol of MDA f orm ed/
mg protein.

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
33
Es tim at i on o f T ota l T hio l:
Total thiol was estimated b y Ell man’s method ( 22).
Assa y mixture con sisted of phosphate bu ffe r (1M,
pH-7 .4), 50 µl serum and 50 µl Ellman’s reagent
[5 , 5’-dithiobis -(2- n itr oben zoi c ac id) (DTN B)] and
incubated in dark for 30 min at r oom temperature.
The thiols interac ted with DT NB, form ing a hig hly
col oured anio n wi t h max i m um peak at 4 12 n m ,
abso rbance were r ecorded at 412 nm.
Es tima tio n o f La cta te:
Calorimetric determination of LA in human plasma
was e s ti mat e d b y t he m et h o d o f Bar k er an d
Summerson (23). Lact ic acid was conver ted into
acet aldehyde by reacting with concentrated sulfuric
acid, and th e ac etaldehyde was measured by its
colo u r r ea c tion wit h p - hy d r ox yd i p h en yl i n t he
pres ence of c upric ions . The colour was read in a
photoelectric calorimeter at 560 nm.
St atis tic a l a n alysi s
Statistical analys es were computed b y us ing t he
Statistical Pac kage for the Social Sciences (SPSS)
vers ion 21.0 (SPSS Incorp, United States). L evel
of significance for all analyses was set at p<0.05.
Data were expressed as mean±standard deviation
(S.D).
All data were exam ined for the normality by using
Shapiro-W ilk Test of norm ality. Repeated me asures
analysis of variance (ANOVA) was use d to compare
the different physiological variables over time among
the t h r e e s upplemental tria l s . These variabl e s
included heart rate, mean skin body te mperatur e,
plasma glucose, plasma lactat e, plasma free fatty
acid and plasm a ins ulin. Bonferroni adjus tment for
mu l t i p l e c omp a r i s o ns w a s u s ed t o l o c at e t he
differences when r epeated mea sures analysis of
varian ce revealed a significant main eff ect of time.
RPE and fluid sens ation sca le wer e analysed by
using Kruskal-W a llis test.
Two tail paired t -test was conducted to test the
significanc e of diff erenc e between mea n values of
pre- and post-trial data.
Results
Physic a l a n d p h ys i o l o gical pa r a m eter s o f the
subjects have been tabulated in Table I that indicated
that the participants of the present study were within
the normal r ange of BMI. There was no significant
alte r a tion i n r oom t e mper a t ur e (30 . 0 ± 0.8 9 º C ,
30 .5±1 .38ºC a nd 2 9.5± 1.87 ºC, res pectively) and
rel a t i ve humidity (67.67±5.09, 6 7 .5±6.63% a n d
67.83±8.3%, respectively) among the three experimental
trials, i.e. PL t rial (PL), h oney supplementation trial
with 30 g of honey (H30) and honey supplementation
trial with 60 g of honey ( H60), resp ectively.
Running time to exhaustion was significantly (p<0.05)
diff erent between the PL and H30 trial and PL and
H60 trial and also between the trial s with two d oses
of hon ey, i.e., H30 and H60 trials respectively (Fig. 3).
TABL E I : Physica l pa ra meters o f the su bjec ts (n=1 2).
Age ( y r s) 22 .5± 0.8 4
Body mas s (kg ) 60± 8 .34
Body he ig ht (cm ) 167.4± 0.04
BMI (kg. m– 2) 21.36±2. 37
Rest ing h ea rt rate (beat s. min–1) 7 1 ± 1 .24
HRma x (beat s. min–1) 197.5 ± 0.8 4
Syst ol ic b lo od press ur e (m m Hg ) 118±7 . 44
Diastoli c blood pre ssure (mm Hg) 78± 9 .57
HRma x = Maxim um heart rate, BMI = Body Mas s Ind ex.
Fig. 3 : Endurance time in different time trials.
*p<0.05 when compared H30 trial with PL trial.
#p<0.05 when compared between H30 and H60 trials.
¥p<0.05 when compared H60 trial with PL trial.
*
#, ¥

3 4 Bhattacharjee and Bandyopadhyay Indian J Phys io l Ph arma col 20 20; 64(1)
HR increased signif icantly (p<0.05), over time in the
same experimental trial co mpared to the respective
rest ing value (T able II). All the work ing HR of H60
trial was significan tly (p<0.05) higher than th e PL
trial. H o w e v e r , work i n g H R had no signi f ic a n t
diff erenc e between H3 0 and H60 trials, except at
90t h m in and the time of exhaustion. H30 trial showed
significant (p<0. 05) difference in working heart rate
at 5th , 20th , 40th , 50th , 60th and 80th min in comparison
to the PL trial. However, the pr e-exercise heart rate,
i.e., the heart just at the beg inning of ea ch exercise
trial did not show any significant difference between
the trials.
Changes in blood paramet ers ha ve been p resented
in Table III. Two way repeated measure ANOVA
depicted significant difference in post-exercise values
of plasma lac tate, blo od glucose, free fatty acids,
insulin, superoxide dism utase, catalase, total thiol
and lipi d peroxidation not o nly between the H30
and H60 trials bu t also between PL trial and two
doses of honey trials. However, p re-ex ercise values
did not s how any inter-trial variation. Pre- and post-
exerc ise values of all these param ete rs showed
significant (p<0.05) dif ference in all the t hree trials
(Table III).
Subjects expressed their conditions on a numeric al
scal e (RPE by Borg’s Scale) to indicate the f atigue
level (Table IV). RPE increased significantly (p<0.05)
from 10th m in of exercise to end tim e of exhaus tion
in PL, H30 and H60 trials, but no significant var iation
was fo und in this parameter when compared among
the three experime ntal trials.
TABL E II : Exercise h ea rt rates (bea ts. min– 1) in three di fferent experime nt al t ri al s (n=1 2).
Exercise Duration (min)
Supple-
mentation 0 5 10 20 30 40 50 60 70 80 90 At
Exhaustion
PL 72.66 116.66 148 162.67 173.33 178.67 186.5 190.67 196.67 200.83 210.33 222.16
±1.97 ±1.50 ±4.05 ±2.42 ±4.32 ±2.73 ±1.87 ±2.16 ±3.98 ±2.64 ±2.16 ±1.33
H30 73.33 114 144.5 159.5 171.33 174.33 184.5 188 193.67 198.17 208.17 220
±1.50 ±2.6 ±4.85 ±2.17* ±3.83 ±3.27* ±1.64* ±2.28* ±3.4 ±1.72* ±2.23 ±1.09*
H60 72.17 110.83 141 157.67 169.33 172 183 185.67 191.33 196.17 205 225.5
±1.47 ±3.6¥±4.29¥±4.23¥±3.88¥±4.56¥±2.19¥±3.01¥±3.67¥±1.72¥±1.1#¥ ±1.38#¥
Values we re expr es se d as M ean± SD ; PL , Placebo; H 30, Ho ne y 30 g; H 60, Ho ne y 60 g.
*p<0.05 when com pare d H 30 t ri al with PL trial.
¥p<0.05 when co mp ar ed H6 0 trial with PL trial.
#p<0.05 wh en com pa red be tw een H30 an d H6 0 trials.
TABLE III : Values of d if feren t blood pa ram et ers d uring t he
exp er im ental trials (n=12).
Paramet ers Trial Pre exercise Post exercise
Plasma Glucose (mg/dl) PL 92.83± 1.94 80±1.4$
H30 93.17± 1.47 90±2.1*$
H60 93.83± 1.17 92.5±1.64#¥$
Plasma insulin (µU/ml) PL 7.22 ± 0.25 1.52± 0.17$
H30 7.25± 0.24 1.92±0.03*$
H60 7.33± 0.14 2.50±0.16#¥$
Plasma Lactate (mmol/L) PL 1.48 ± 0.1 5.28±0.25$
H30 1.42± 0.1 4.55±0.13*$
H60 1.39± 0.09 4.38±0.14#¥$
Plasma Free Fatty PL 0.55 ± 0.03 1.07±0.01$
Acid (mmol/L) H30 0.53 ± 0.02 0.99± 0.03*$
H60 0.52± 0.01 0.76±0.03#¥$
Lipid peroxidation PL 1.56 ±0.01 2.05± 0.12$
(µmol/L) H30 1.57± 0.02 1.26±0.01*$
H60 1.54± 0.01 0.99±0.02#¥$
Catalase (µmol of H2O2/ PL 72.24 ± 0.89 77.22±1.21$
min/mg of protein) H30 71.61± 1.8 79.24± 1.52 *$
H60 70.25± 1.21 82.4±1.47#¥$
Superoxide dismutase PL 16.76 ± 1.58 21.39±1.74$
(nmol/mg of protein) H30 16.3± 1.82 24.53±1.68*$
H60 15.32± 1.42 27.67±1.32#¥$
Total Thiol (µmol/L) PL 15.96 ± 1.64 20.20±1.51$
H30 15.2± 1.61 23.46 ± 1.6*$
H60 14.45± 1.22 26.05±1.53#¥$
Va lu es were e xpress ed as M ea n±SD.
PL= Pl ac eb o; H30 = Ho ne y t rial with 30 g dose ; H6 0 =
Honey trial with 6 0 g dos e.
*p<0.05 when co mpar ed H30 trial wi th PL t ri al
#p<0.05 wh en c om pared between H30 and H60 tr ials
¥p<0.05 when co mp ar ed H6 0 trial with PL trial.
$p< 0 . 05 wh en co m p are d w ith th e c orr e s pon d ing p r e–
exer cis e v a lu e
Discussion
In the present study, acute supplementation of honey
in two different doses significantly (p<0.05 ) improved
the endurance performance comp ared to PL trial.

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
35
Endurance time to exhaustion was significantly higher
in H30 and H60 tr ials in comparison to PL trial and
also between H30 and H60 trials (Fig. 3). This finding
was similar with previous study where chronic honey
supplem entat ion was foun d to be beneficia l f o r
exer cise performance as it improved power output
and blood glucos e level during endur ance trials . In
one study, effect of low glycemic index (honey; GI
= 35) and high G I (dextrose; GI = 100) carbohydrate
feed ings during a s imulated 6 4-k m cycling time trial
(TT) showed improvem ent in tim e and power output
ov e r the last 16 km of a 64-km simula t ed TT
regardless of glycemic index (6). T his was probably
due to the f act that honey consists of mainly fructose
along with glucose which helps to maintain blood
glucose level during endurance exercise and help to
spar e muscle glycogen (3) .
Exercising heart rates of each tria l i n c r e a s e d
significantly (p<0.05) compared to the cor responding
rest ing value to meet the increased dem and of the
body during physical exercise. The working heart
rates were significantly (p<0.05) lower in the H60
trial than the PL trial. H 30 trial also had significantly
(p<0.05) lower values of working heart rates than the
PL trial at 0th, 20th, 40th , 50th, 60th , 80th min and at
the time of exhaustion. However, working heart rates
had no significant variation between H60 and PL trials.
T h e se f in din g s in d i cat e d t ha t hon e y e xe r t ed
significant beneficial effects on heart r ate during
endurance trials which is in agreement with the earlier
findings of Kreider (1). The components of honey i.e.
fr uctose a nd glucose along w ith the anti-ox ida nt
en zymes w e re po s s i bly the re a s on be h i nd th i s
lowering of heart rate.
In this inves tigat ion, post-exercis e blood glucose
level was increased after ingestion of honey in both
the dos es than PL. Existence of sign ificantly higher
(p<0.05) post exercise blood glucose in H30 and
H60 trials compared with that of PL trial was in
agree m e nt w i t h e a rli er i nv e s ti g at io n s ( 2, 2 4 ) .
Beneficial effects of consuming a high-maltodextrin,
low-fructose beverage (Mf) over normal carbohydrate-
rich diet on en ergy exchange, substrate m etabolism,
and per formance were als o esta blished (25). There
was statistically significant glycogen sparing effect
along with super-compens atory eff ect within 24 h of
recovery following Mf supplementation. Performance
impr oved to a better extent for 1h tim e trial with
inges tion of a 2:1 gluc ose-fructose mixture than with
ingestion of gluc ose only ( 26). In f act, the oxidation
rate of a 2:1 mixture of glucos e and fructose was
higher than glucose alone and end ogenous CHO
oxidatio n was lower ( 27). It was quite eviden t as the
cons tituents of h o ney a r e m a i nly f r u c t o se and
glucose, it prevented the decre ase in blood glucose
level during exercise in H30 and H60 trial than PL
trial (1).
Post exercise serum insulin level was sig nific antly
higher (p<0.05) in honey trials than PL trial. H30
and H 60 t rials a lso sho wed sig nificant (p<0.05)
difference in post exercise insulin level. Carbohydrate
feeding leads to elevated level of blood glucose, which
in tur n increases the syn thesis of ins ulin by the
pancreatic beta cells for adjusting the excess glucose
in b lood. The greater insulin level might be due to
better regulatory mechanism of blood g lucos e by
honey supp lementation (28).
A signif icant (p<0.05) increase in plasma lactate
concentration was noted in all the three experimental
trials. PL trial showe d gr eater post-exercise lac tate
produc tion than honey tr ials (p<0.05). This increase
in post -exercise lactic acid might be either due to
increas e d l a c t a te p r o d u ct i o n o r i t s d e c r ea s ed
clearance or com bination of both or due to increased
contribut io n of n o n - o x i d a t i ve e n e r g y path ways
impo sed by insufficient blood flo w to the exercis ing
muscles (11).
TABL E I V : Rate of perc ei ve d exertion (RPE) of the subject s
(n=12) dur in g e xercis e i n d if ferent tri al s.
Exercise Duration (min) PL H30 H60
10 10.9±3.2 10.5±2.7 10.3±2.2
20 11.4±2.4 11.1±2.3 10.7±2.1
30 11.9±2.6 11.4±2.5 11.2±2.5
40 12.5±2.8 12.3±2.4 11.8±2.6
50 13.1±2.7 12.4±2.8 12.1±2.4
60 13.9±2.9 13.6±2.6 13.4±2.7
70 14.7±1.9 14.5±1.2 14.3±2.3
80 16.6±2.3 16.4±2.1 16.2±2.2
90 18.8±1.2 17.8±1.0 17.5±1.1
At exhaustion 19.8±0.5* 19.3±0.5* 19.2±0.4*
Va lu es were e xpress ed as M ea n±SD.
PL = Pl ac ebo t ri al ; H30 = Ho ne y trial wit h 30 g dos e.
H60 = Hone y t rial with 60 g do se .
*p<0.05 when com pared with 10th min value in the s ame trial.

3 6 Bhattacharjee and Bandyopadhyay Indian J Phys io l Ph arma col 20 20; 64(1)
Plasm a f ree fat t y ac i d i n c re as ed s i gn i f ic an tl y
(p<0.05) a t the end of the exercise trials comp ared
to their corresponding resting values. Plasm a free
fatt y acid is the most im portant energ y sourc e to
sus t a in pr o longed endur a n c e act ivity (29) . The
increase of FFA was probably due to the av ailability
of the s ubstrate (fat) for its utilization as the m ajor
energy s ource during the endurance running trials.
Sign i f ic a n t ly (p<0 . 0 5 ) l o w e r post-exercise FFA
con c entrat i ons we re ob s e rved in H3 0 and H60
trials than PL trial. Between honey trials, H60 causes
less increase in post ex ercise FFA concentration
th a n H30 trial . Other study als o ind i c ated t hat
carbohydrate sup ple m entation meets the ener gy
demand during exercise by sparing muscle glycogen
and body l i p ids (2). I n crease in s e rum insulin
afte r carbohydrat e feeding in H 30 and H60 trials
migh t be a reason beh ind this d ecrease in plasma
FFA.
Honey has free-rad ical scavengi ng capacity. Honey
supplemen tatio n depicted a s ignif i cant (p<0. 05)
increa se in oxidat ive stress markers (SOD and total
thiol) and a significant (p<0.05) decreas e in LPO
level in H30 and H60 trials compa red to the PL trial.
Significant (p<0.05) variation in catalase activity was
obse rved not only betwe en H30 and H60 and trials
but also bet ween PL trial and honey trial s with two
different doses. Similar changes were noted in SOD,
catalase and total thiol. Honey is made up of various
phenolic an d non-phenolic anti- oxidant components
(e.g. glucose oxidase, vit amin C, vitamin E, be ta
caro tene etc.) which helps to n eutrali se the fr ee
radicals generate d during endurance exercise and
thus the blood levels of oxidative str ess markers
increased (30) .
A significant increase (p<0.05) in RPE was observed
between pre -exercise (0 min) value and end time to
exha ustion value in all the three t rials . But RPE did
not show any significant (p<0.05) difference between
the three trials. Although statistically insignificant,
the end time to exhaustion valu e of RPE was greater
in PL trial than H30 and H60 tr ials . This reduction of
RPE af t er hone y feeding m i g ht be l i n k e d with
incre ased oxidation of carbohydrate, well maintained
blood glucose as well as elevated levels of ser um
insulin (11). But the exact reason behind having the
existenc e of insignif icant dif ference in RPE between
any of the trials is not known.
Ther efo re s ignif icant changes in all the param eters
were observed in the present study during both the
honey trials in comparison to PL trial. Moreover, H60
proved to be better than H30 in all the aspects. So,
it c an be hypothesized that the acti ve ingredien ts in
honey were sufficiently abso rbed during the trials
and elicited positive eff ects towards performance
enhancement by means of increasing the endurance
time.
In conclusion, acute supplem entation of dif fer ent
do ses o f hon ey one hour prio r to the exerc ise
perf orm ance exerte d significant effect than tha t of
PL on the physiological parameters measured during
the e n durance runn i ng performance in h ealthy
recr eationa l run ners probably by maintainin g blo od
glucose level during endurance exerc ise as well as
by reducing oxidative stress in healthy individuals.
T h i s st u dy als o s ign i f ied t ha t 60 g o f ho n e y
supp lementation was m ore potent as ergogenic aid
than 30 g of honey supplementation. Hence, honey
can be a prospect ive ergoge nic aid for end urance
athletes.
Pr act i ca l / S o ci a l a ppl i cation of t h e st ud y:
Endurance exercise p erformance can be increased
by c a r bo hyd rat e f eed i n g . Ho n e y i s a n a t ural
carbohyd r a te s o u r c e, a com b i n a t i o n o f m a i n l y
fructose and glucose which helps to maintain blood
glucose lev el required for strenu ous e xercise. Due
to its low glycemic index, honey helps in susta ined
releas e of glucose in blood stream , sparing stored
muscle glycogen. It c onsists of various antioxidant
enzym es which can com bat oxidative stress. H ence
it can be u sed as energy dr ink prior to exercise for
optimu m exe rcise performanc e.
Acknowledgements
Authors ar e indebted to all the p artic ipants for t heir
whole hear ted suppor t to com plete the study.

Indian J Phys io l Ph arma col 2020; 64(1)
Effects of Acute Supplementation of Honey on Endurance Performance
37
References
1. Krie der RB, R as musse n C J, L an ca ster SL, K er ks ick C ,
Greenwo od M. Honey: An alt ernati ve sports gel . Strength
Cond J 20 02; 24(1): 50 –5 1.
2. Jeuk endrup AE. Carbohyd ra te int ak e during exer ci se and
perfor mance. N ut r 200 4; 20 (7 -8 ): 669–677.
3. Bogdanov S , Ju rendic T, Si eber R, Gallma nn P. Ho ne y for
nu t rit i on a n d he alt h: a r evi ew. J A m Col l Nut r 200 8 ;
27(6): 6 77–6 89.
4. Khalil M, S ul ai man S , B oukraa L. A nt ioxida nt Prop erti es of
Honey and its ro le in Preven ting Healt h Di so rd er . O pe n
Nutrac eu tic als J 20 10; 3 : 6 –1 6.
5. Bert oncelj J, Dobers ek U, Jam ni k M, Golob T. Evalu at io n
of the phenolic content, antioxidan t activity and colour of
Slovenia n hone y. F oo d Chem 2 00 7; 1 05 (2 ): 8 22–8 28 .
6. Earnest C, Lanca st er S, Rasm u ss en C , et al. Low versus
hi gh g lyc e mic i nde x m eals c a rb ohy dra te g el in ges tio n
during sim ulat ed 64 km cy cl in g time tria l perf or mance. J
Stre ngth C on d Res 2004; 18(3): 46 6– 47 2.
7. Bu c eko va M, V ala chov a I, K o hu t ova L, P r och azk a E ,
Kl aud iny J, M aj t an J . “H one ybe e g luc ose o xi das e—i t s
ex p r e s si on i n h o n e yb e e w o r ke r s a n d c o m pa ra t i ve
analyses of it s c on tent an d H 2O2-mediated antibac te ri al
ac tiv ity i n na tu ral h oneys ”. Natu r wi s sen sch aft en 2 014 ;
101(8) : 661–670.
8. Jackson C GR . Nutri ti on f or the recreatio nal Athletes. T aylor
and Fran ci s Limit ed P ub li cation, CR C Pre ss , Gree ley Inc ;
1995.
9. Dupont W D, Plum mer W D. Jr: Power and Sampl e Size
Ca l cula t ion s f or St u d ies I n vol v ing L i nea r R egr essi o n.
Cont ro l C lin T ri als 19 98; 19 (6 ): 589–6 01 .
10. Shenoy VP, Ballal M, Shivananda P G, Bairy I . Hon ey as
an antimi cr ob ial age nt agai ns t Pseudomonas aer ugin os a
isolat ed fro m infec te d wo unds . J Global In fe ct Dis 20 12 ;
4(2): 1 02–105.
11. Ping FW D, K eong CC, Bandyopadhya y A. Eff ec ts of ac ute
suppleme nt ation of Panax ginse ng on endu rance running
in a hot and h um id envi ro nm ent. Ind ia n J Med Res 2011;
133(1): 9 6–10 2.
12. Murray R. F lu id re placeme nt : T he America n C ol lege of
Sports Medi cine Position Stand . Sport Sc i Ex ch 1996; 9:
1– 5.
13. William MH. Nutr it ion f or hea lt h, fi tn es s a nd sport, Boston:
Mc GrawH il l; 2002.
14. Lo wry O H, R o seb rou g h N J , Far r AL a n d R anda ll RJ .
Protei n measureme nt with the foli n phenol reag en t. J Bi ol
Chem 1 95 1; 19 3( 1) : 2 65 –2 75 .
15. Kunst A, D raeger B, Zi egenhorn J. In: Bergme yer. Methods
of E n z ym a ti c A n a ly s is , 3 r d e d. M e ta b ol i te s 1 :
Carbohydrates. 19 84 ; 6 : 1 63 –1 72 .
16. Tiet z NW. ed. Cli ni cal Guide to Laboratory Tests , 4th ed.
WB Saunders Com pa ny , Ph il ad el phia . 2006; 444–451.
17. Sapin R, Galudec V L, Gas ser F, Pi ng et M and Grucke r
D. Elec sys Insulin Assa y: Free Ins ulin Det er mination and
the Ab senc e of Cr os s-Reactivit y wi th Insu li n Lispro. Cli n
Chem 20 01; 47(3): 60 2– 60 5.
18. Kwo n DY and Rhee JS. A Simple and Rapid Col or im etric
Method for D et ermin at i on of free f atty aci ds for li pase
assa y. J A m Oi l Chem S oc 19 86 ; 63(1): 89 –9 2.
19. Mar klun d S an d Marklund G. Invol ve me nt o f th e supe ro xide
an i on ra d ical i n t he au t oxi d ati o n o f pyr o gal l ol a n d a
c o n v e n i e n t ass a y f or s up e r o x i d e di sm ut a s e . E u r J
Bi oc he m 1 97 4; 47 (3 ): 46 9–47 4.
20. Cl a i born e A . C a tala s e a c tivi t y , in CRC H a n dbo o k o f
Meth od s f or O xy gen R ad ica l Rese ar ch, ed ite d by R A
Green Wald (C RC Pre ss: Boc a Raton , FL) 1985; 283 .
21. Wright JR, Colby HD a nd Mi les PR . Cy tosolic facto rs that
affe ct microsoma l l ip id pe ro xidati on in lung and liver. Arch
Bioc he m Bioph ys 19 81 ; 2 06 (2 ): 2 96 –304 .
22. Ellm an G L. T i ssue sul fhyd r y l g r oups. A r ch B iochem
Biophy s. 1 95 9; 8 2( 1): 70–77.
23. B a r k er S B a n d S u mm er s on W H. T h e c o l o r im et r ic
determ in at io n o f lac ti c acid in biological ma te ri al . J Biol
Chem 19 41 ; 138(2): 535–554.
24. Wallis GA, Yeo SE , B lannin AK, Jeukendrup AE. Do se-
re spons e e ffe ct s of i ng est ed car bohyd ra te o n e xe rci se
meta bo li sm in women. Med Sci Spo rt s Exe rc 2007; 39 (1 ):
131–138.
25. Brouns F, Sa ris WHM, Beck ers E, et al. Metabolic c hanges
in d u c ed by s us ta i n ed e xh a u s ti v e c y cl i n g an d d ie t
mani pu la ti on . In t J Sport s M ed 1989; 1 0( 1): S 49 –S 62 .
26. Currell and J euke nd ru p CK and Jeuk en drup AE. S uper io r
en d u r an c e p e rf o rm a nc e w i t h i n g e s ti on of m ul ti pl e
transportabl e car bohydrates . Med Sci Sports Exerc 2008;
40(2): 2 75–2 81.
27. Je ntj ens R L, M ose ley L, War ing RH, Har din g L K and
Jeuke ndrup A E. O xidat ion of combined ingest io n of gl ucos e
and fru ct os e duri ng exer ci se. J Appl Phy siol 2004; 96(4):
1277–1284.
28. Al Wai li NS . N at ural honey l ow ers p lasma gl ucose , C -
reactive protein, ho mocysteine, and blood lipid s in hea lthy,
di a b e ti c a n d h y pe r li p i de m ic s u bj e c ts : c om pa r is o n
wit h dextrose and sucrose. J Med Food 2004; 7(1) : 100–
10 7.
29. Jeff re y F, Ho rowi tz and Samu el K lein. L ip id m et abol ism
du ri ng e nd ura nce exer c is e 1– 3, A m J Cli n Nu tr 200 0;
72(2): 55 8S –563S .
30. Ghazali W SW, Mohamed M, Sulaim an SA, Az iz AA an d
Yu s o ff HM . Tua l ang h one y s upp l emen t ati o n impr ove s
oxi da tive stress sta tu s amo ng c hr on ic smoke rs . J Toxi co l
Envi ro n Chem 2 01 5; 97 (8 ): 1 01 7– 10 24 .