ORIGINAL ARTICLE

Determinants of muscle metaboreflex and involvementof baroreflex in boys and young men

Konstantina Dipla • Stavros Papadopoulos •

Andreas Zafeiridis • Antonios Kyparos •

Michalis G. Nikolaidis • Ioannis S. Vrabas

Received: 14 January 2012 / Accepted: 3 September 2012 / Published online: 15 September 2012

� Springer-Verlag 2012

Abstract This study aimed to assess the arterial pressure

(AP) determinants during the muscle metaboreflex in boys

and men and to investigate the contribution of baroreflex

and sympathovagal function to the metaboreflex-induced

responses. Fourteen pre-adolescent boys and 13 men per-

formed a protocol involving: baseline, isometric handgrip

exercise, circulatory occlusion, and recovery. The same

protocol was repeated without occlusion. During baseline,

boys had lower beat-to-beat AP, higher heart rate (HR),

and lower low/high frequency HR variability. During

exercise, a parasympathetic withdrawal was evident in both

groups. In adults, HR was the key contributor to the

pressure response, with no changes in stroke volume,

whereas in boys, the lower HR increase was counterbal-

anced by an increase in stroke volume, resulting in similar

relative increases in AP in both groups. In recovery, boys

exhibited a faster rate of HR-decay, rapid vagal reactiva-

tion, and greater decrease in TPR than men. An overshoot

in baroreceptor sensitivity was observed in men. The iso-

lated metaboreflex resulted in a similar AP elevation in

both age groups (by *15 mmHg), and attenuated sponta-

neous baroreceptor sensitivity. However, during the me-

taboreflex, pre-adolescent males exhibited a lower increase

in peripheral resistance and a greater bradycardic response

than adults, and a fast restoration of vagal activity to non-

occlusion levels. During metaboreflex, boys were capable

of eliciting a pressure response similar to the one elicited

by men; however, the interplay of the mechanisms under-

lying the rise in AP differed between the two groups with

the vagal contribution being greater in the younger

participants.

Keywords Isometric exercise � Children � Metaboreflex �Baroreflex � Stroke volume � Parasympathetic �Blood pressure � Heart rate � Post-exercise recovery

Introduction

The blood pressure adjustments to daily perturbations are

regulated by the interplay of total peripheral resistance

(TPR) and cardiac output, which in turn are under the

control of the autonomic nervous system (Mitchell et al.

1983). During exercise, the ‘‘central command’’, the

‘‘exercise pressor’’ reflex, and baroreceptors integrate TPR

and cardiac output, resulting in an effective elevation of

blood pressure (Mitchell et al. 1983; Ogoh et al. 2007;

O’Leary 1996; Rowell and O’Leary 1990). More specifi-

cally, ‘‘central command’’ has been considered as a feed-

forward neural drive originating in higher brain centers and

is involved in recruiting motor units and activating car-

diovascular control centers within the brainstem to adjust

sympathetic and parasympathetic nerve activity (Iellamo

et al. 1997). The ‘‘exercise pressor reflex’’, a feedback

drive, transmits signals from the contracting skeletal

muscle via group III fibers (sensitive to changes in the

mechanical properties, the ‘‘mechanoreflex’’) and group IV

fibers (sensitive to increased metabolite concentration, the

‘‘metaboreflex’’) (Mitchell et al. 1983; Kaufman et al.

1983; Iellamo 2001). In addition, the arterial baroreflex

exerts its function through afferent fibers arising in the

Communicated by Massimo Pagani.

K. Dipla (&) � S. Papadopoulos � A. Zafeiridis � A. Kyparos �M. G. Nikolaidis � I. S. Vrabas

Exercise Physiology and Biochemistry Laboratory,

Department of Physical Education and Sport Sciences at Serres,

Aristotle University of Thessaloniki, Agios Ioannis,

62110 Serres, Greece

e-mail: kdipla@phed-sr.auth.gr

123

Eur J Appl Physiol (2013) 113:827–838

DOI 10.1007/s00421-012-2493-7

carotid sinus and the aortic arch and is responsible for

instantaneous adjustments in blood pressure (Rowell and

O’Leary 1990; O’Leary 1996).

During growth and maturation, the autonomic nervous

system undergoes changes (Finley and Nugent 1995;

Pikkujamsa et al. 1999; Lenard et al. 2004). From 5 to

10 years of age, a decline over time in sympathetic and

possibly a slight decline in parasympathetic activity has

been reported (Finley et al. 1987). Indicators of sympa-

thetic modulation have also been reported lower in pread-

olescents compared with adolescents (Tanaka et al. 2000).

In addition, lower resting cardiovagal baroreflex sensitivity

(BRS) has been reported in children compared with ado-

lescents and young adults, suggesting that baroreceptor

regulation improves from early childhood to adolescence

(Lenard et al. 2004).

The ability for prompt adjustments in hemodynamic

regulation might not be apparent at resting measurements

(Piepoli et al. 2008). Therefore, physiological maneuvers

(such as isometric exercise) and pharmacological approa-

ches (such as atropine) have become popular for studying

the autonomic regulation in adults (Tulppo et al. 2001;

Yamamoto et al. 1995; Castiglioni et al. 2011; Parati et al.

2000; Joyner and Dietz 2003). However, most studies that

investigated the effects of maturation of the autonomic

system were performed under resting conditions. Recently,

Goulopoulou, Fernhall, and Kanaley (Goulopoulou et al.

2010) compared the hemodynamic responses to isometric

exercise in children and in adults and they reported that

children exhibited a lower exercise pressure response

compared with adults. However, in the aforementioned

study the exercise pressor reflex was examined as a whole,

involving all peripheral reflexes. Therefore, whether the

contribution of each of the peripheral reflexes (metabore-

flex or the mechanoreflex) is different between children

and adults has not been clarified. Turley (Turley 2005)

examined the arterial pressure (AP) responses during

occlusion and reported similar responses in children and

adults. In that study, the circulatory responses were not

performed on a beat-by-beat basis and the mechanisms

involved in the maintenance of AP during the metaboreflex

were not examined. In addition, whether the interaction of

baroreceptors’ function with the muscle metaboreflex is

different in children and in adults has not been investi-

gated. Therefore, the purpose of this study was to examine

(1) the arterial pressure response during the metaboreflex in

boys and in adult males and the hemodynamic variables

that determine this response and (2) the involvement of

baroreflex and parasympathetic function to the metabore-

flex response in the two groups. Based on the previous

reports (Goulopoulou et al. 2010), we hypothesized

that boys would elicit a lower arterial pressure response

during the isolated metaboreflex compared with adults.

In addition, we anticipated that boys would demonstrate a

higher baroreflex and parasympathetic involvement to the

metaboreflex response than adults.

Materials and methods

Participants

Twenty-eight participants were recruited for the study. The

sample comprised of 14 healthy lean boys (aged 11.7 ±

0.3 years; Tanner stage 1–2; body mass 43.0 ± 2.0 kg;

body mass index, BMI 18.69 ± 0.34 kg/m2) and 13 heal-

thy lean men (aged 24.8 ± 1.4 years; body mass 77.2 ±

3.2 kg, BMI 24.10 ± 0.74 kg/m2).

Participants with known major diseases, such as car-

diovascular, respiratory, metabolic, or renal disease as well

as participants who were under medication were excluded

from the study. The study was conducted in conformity

with the Declaration of Helsinki. The study protocol was

approved by the institutional review board committee.

Detailed information on the study benefits, risks, and pro-

cedures was provided in writing to the adult participants,

the children, and their parents. All adult participants and

children’s parents signed the written informed consent

form and completed a medical questionnaire. The partici-

pants were asked to follow their normal daily diet, to have

sufficient rest the night before the study, to abstain from

intense exercise activity for at least 24 h before the study,

and not to consume any food, water, or tobacco products at

least 3 h before the test.

Testing procedures and instrumentation

Participants arrived in the laboratory and were habituated

to the experimental equipment and testing procedures.

Height was measured using an accurate stadiometer and

body mass using a weighting scale (SECA, Hamburg,

Germany). BMI (in kg/m2) was calculated. Pubescent stage

was determined according to pubic hair and gonadal

development. A portable hydraulic dynamometer (5,030 J,

Sammons Preston, Chicago, IL), was used for assessing the

maximal voluntary contraction (MVC). The test was per-

formed in the sitting position with the elbow flexed at 90�,

with the forearm and wrist set in neutral position. The

participant performed three maximal isometric handgrip

(HG) trials with the dominant hand intercepted by a 60-s

resting period between each measurement. The participants

were instructed to squeeze the dynamometer, as hard as

possible. The highest reading produced was considered as

the participant’s MVC; this value was then used to derive

the 30 % MVC used during the experimental condition (i.e.

MVC 9 0.3). Then, the participant rested for about 15 min

828 Eur J Appl Physiol (2013) 113:827–838

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while the preparation of the ECG equipment (MP150,

Biopac, Santa Barbara, California, USA) and finger pho-

toplethysmography, Finometer, Finapres Medical Systems,

Amsterdam, The Netherlands) apparatus took place. Con-

tinuous (beat-to-beat) arterial pressure was obtained from

the middle finger of the non-dominant hand, supported at

the heart level. Before the initiation of the protocol, bra-

chial artery blood pressure (BP) was assessed by an auto-

mated sphygmomanometer (Omron, Matsusaka, Japan) in

the dominant arm to verify Finapres measurements of

absolute BP. The Finapres system has been shown to

provide accurate estimates of intra-arterial pressure (Parati

et al. 1989), tracking changes in BP at rest and during

exercise. An inflatable cuff for arterial occlusion was fixed

to the upper arm on the dominant hand.

Participants performed two experimental protocols in a

random order: (1) a Post-Exercise Circulatory Occlusion

(PECO) protocol and (2) a control, non-PECO, protocol.

The PECO protocol involved a 3-min rest period (baseline),

followed by a 3-min exercise period. During the exercise

period, the participant was asked to maintain force output to

the predetermined percentage (30 %) of his MVC. Visual

feedback was provided during the exercise period and the

participant was constantly reminded to relax any other

muscles not involved in the HG exercise. Ten seconds before

the cessation of the HG exercise period, forearm arterial

blood flow occlusion was rapidly initiated. The pressure in

the cuff of the exercising arm was maintained at suprasy-

stolic levels (50 mmHg above peak exercise arm systolic

BP) for 3 min after the cessation of exercise (Crisafulli et al.

2003), to prevent removal of the muscle metabolites and

examine the hemodynamic responses induced only by

metaboreceptors’ stimulation. A three-min recovery period

followed. After a 60-min recovery, the participant per-

formed the same protocol without circulatory occlusion

(non-PECO), involving 3 min of rest (baseline), 3 min of

HG exercise, 3 min without circulatory occlusion (-occlu-

sion), and 3 min of quiet recovery. During the test, partici-

pants were instructed not to talk or perform any unnecessary

movements. The participants were continuously instructed

to maintain their initial breathing rate (Nishiyasu et al.

1994), to avoid breath holding and the Valsalva maneuver

throughout the experiment. The breathing frequency was not

fixed through a metronome due to the finding in our pilot and

previously published studies (Tanaka et al. 1998) that chil-

dren have difficulties performing metronome rhythm

breathing successfully over several minutes. The testing

session was aborted in one adult who complained of dis-

comfort during the second minute of occlusion and did not

relax even when the pressure in the cuff was reduced by

15 mmHg. This subject was not included in the study.

Systolic and diastolic arterial blood pressure and heart

rate (HR) were continuously recorded; mean arterial

pressure (MAP) was calculated using BeatScope software

(version 1.a). Stroke volume (SV) was computed by the

Modelflow method (arterial pressure wave analysis), as

described by Jansen et al. (2001). The Modelflow is a

noninvasive method that has been shown to be a reliable

alternative to invasive thermodilution techniques (Jansen

et al. 2001). The metaboreflex effect on MAP was assessed

as the difference in MAP values between the occlusion and

non-occlusion periods. BRS (ms/mmHg) was assessed by

the sequence method (Iellamo et al. 1994) using the

Beatscope software (Beatscope 1a, Finometer, Finapres

Medical Systems). In brief, episodes of three or more

consecutive beats with upward or downward blood pres-

sure ramps and R–R interval alterations following the same

pattern were identified. These heart rate changes tend to

buffer the blood pressure ramps and are assumed to be

generated by the baroreflex (Iellamo et al. 1994). A linear

regression was applied to each individual sequence. Only

those in which the variance (r2) was [0.85 were accepted.

The final BRS estimate was the average slope of all these

sequences and was considered as the index of BRS (Iell-

amo et al. 1994).

Heart rate variability was assessed in compliance to the

Task Force of the European Society of Cardiology and the

North American Society of Pacing and Electrophysiology

(Task Force of the European Society of Cardiology and the

North American Society of Pacing and Electrophysiology

1996) using continuous ECG recording (MP150, Biopac,

Santa Barbara, CA, USA). The analysis was performed

using the HRV Analysis software 1.1 (kindly provided by

The Biomedical Signal Analysis Group, Department of

Applied Physics, University of Kuopio, Kuopio, Finland;

http://venda.uku.fi/research/biosignal). Data were sampled

at 1,000 Hz and saved for further analysis. R–R interval

series were checked by visual inspection for ectopic beats

or artifacts. All HRV data were analyzed by the same

researcher to eliminate inter-observer variability. Baseline

data for HRV analysis in the frequency domain were reg-

istered over a 3-min period of quiet rest in the sitting

position. The interval tachogram analyzed contained data

segments of 180 s consecutive R–R intervals window. Data

were detrended with a ‘‘smoothness priors trend’’ (after

examination that the spectral components were not signif-

icantly altered) and interpolated at 4 Hz (Tarvainen et al.

2002). The interval series were converted into a 1,024-

point signal in the frequency domain and the power spec-

trum was estimated using non-parametric spectral analyses

(Fast-Fourier transformation) with the Welsh periodogram

(with a 50 % window overlapping) and Hanning window.

The resulting periodogram was integrated over very low

frequency (VLF 0.025–0.04 Hz), low frequency (LF) band

(0.04–0.15 Hz), and high frequency (HF) band (0.15–0.4).

The LF/HF power ratio was calculated as an index of the

Eur J Appl Physiol (2013) 113:827–838 829

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sympathovagal ratio at rest. The VLF band was not con-

sidered in the analysis due to the short-term recording of

the data. In the time domain, the root mean square of

successive differences (RMSSD) between the coupling

intervals of adjacent R–R intervals was computed as an

index of parasympathetic activity. RMSSD was computed

separately for each 3-min period of the protocols (i.e. 180-s

long windows for baseline, HG exercise, ± occlusion,

recovery). In addition, the Poincare plot analysis was used

as a non-linear method that does not require ‘‘stationarity

of data’’ and was used for data analysis during each 180 s

period (baseline, exercise, and recovery) (Tulppo et al.

1996). This method is a scattergram in which each R–R

interval is plotted as a function of the previous RR interval

and provides a graphical representation of cardiovascular

dynamics in an ellipse. The ellipse is fitted onto the line of

identity at 45� to the normal axis. The standard deviation of

the points that are perpendicular to the line of identity,

SD1, describes the short term R–R variability and the

standard deviation along the line of identity, SD2,

describes the long-term RR variability.

Statistical analysis

Data are reported as mean ± SEM. Differences in physical

characteristics and in MVCs between the two groups were

assessed by Student’s t tests for independent samples. For

statistical analysis, the physiological variables derived by

the Finapres were averaged over the 3-min periods. Three-

way ANOVAs (group 9 PECO 9 time) with repeated

measures on ‘‘PECO’’ and ‘‘time’’ were used, followed by

Newman–Keuls post hoc tests. The behavior of the HR

during the first minute of recovery was analyzed using a

second-order exponential decay fitting (MicroCal Origin

Pro 8.0, OriginLab Corp, Northampton, MA, USA) using

the following equation:

y ¼ y0 þ A1 � e� x=tð Þ1 þ A2 � e

� x=tð Þ2 ;

where y0 = Y offset, A = amplitude, t1 and t2 = the time

constants. The exponential fitting procedure splits the data

series in two parts through time domain by fitting two

slopes (A1 and A2 slopes in t1 and t2 periods). Then, a

smoothed curve is fitted through the entire data in a relation

with the two slopes.

Results

Subject characteristics

Boys compared with men had significantly lower

(P \ 0.001) body mass (43.0 ± 2.0 vs. 77.2 ± 3.2 kg,

respectively), body mass index (BMI 18.69 ± 0.34 vs.

24.1 ± 0.74 kg/m2, respectively), MVC (24.9 ± 1.8 vs.

55.4 ± 2.8 kg, respectively), and MVC adjusted per unit of

body mass (0.58 ± 0.03 vs. 0.70 ± 0.04 kg/kg of body

mass, respectively).

Hemodynamic responses and metaboreflex evaluation

Representative raw data for beat-by-beat blood pressure

during the PECO and non-PECO protocols are shown in

Fig. 1 (PECO a and b, non-PECO c and d, in one child and

one adult, respectively). Average data for MAP and HR

during the protocols are presented in Fig. 2 (a and b,

respectively). Boys had a lower MAP and higher HR at rest

and throughout the protocols (PECO and non-PECO,

P \ 0.05; Fig. 2). During HG exercise, an increase

(P \ 0.05) compared with baseline was detected in MAP

and HR in both groups (in both protocols). During the

?occlusion period, MAP remained higher than baseline

(P \ 0.05) and was higher in ?occlusion compared with

-occlusion (P \ 0.05), while HR significantly declined

(P \ 0.05) from HG exercise levels and returned to base-

line levels, in both groups. In men, HR was higher

(P \ 0.05) in ?occlusion compared with -occlusion,

whereas in boys there were no differences between

?occlusion and -occlusion. During recovery, in the PECO

protocol (following the release of cuff pressure) MAP

returned to baseline in adults, whereas it remained higher

than baseline (P \ 0.05) in children. During the same

period in the PECO protocol, HR in men continued to

decline (P \ 0.05) and was significantly lower than the

baseline and the ?occlusion periods, whereas in boys

although HR was significantly lower than baseline, no

significant decreases in HR compared with ?occlusion

were observed. Significant differences were observed at

rest and throughout the protocols in absolute SV and TPR

(Fig. 2c, d, respectively) in boys compared with men.

When the rate of decline in HR during the first min of

post-exercise recovery was explored, children exhibited

faster (P \ 0.05) time constants than men (t1 5.22 ± 1.2

vs. 10.05 ± 1.04 in boys and in men, respectively;

t2 4.88 ± 1.2 vs. 10.4 ± 2.3, in boys and in men, respec-

tively). Representative data and exponential decay fitting

from one child and one adult during the first minute of

recovery are presented in Fig. 3 (a and b, respectively).

The muscle metaboreflex-elicited effect on MAP

was not significantly different between boys and men

(D 15.9 ± 2.1 vs. 15.4 ± 3.9 mmHg, in boys vs. men,

respectively). Next, the percent change from baseline val-

ues in MAP and in the parameters determining MAP (i.e.

HR, SV, and TPR) was calculated (Fig. 4). During HG

exercise, the % change from baseline in MAP was not

significantly different in the PECO protocol compared with

830 Eur J Appl Physiol (2013) 113:827–838

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the non-PECO protocol as well as between the two age-

groups (Fig. 4a). During the ±occlusion period, the change

in MAP was higher in the PECO compared with the non-

PECO protocol, within each group. During the recovery

period, the change in MAP in the PECO protocol (i.e.

immediately after releasing occlusion pressure) was higher

(P = 0.05) in boys than in men.

The change (%) from baseline in HR exhibited a dif-

ferential pattern during the time course of the protocols in

children compared with men (Fig. 4b). During HG, boys

exhibited a lower % increase in HR (P \ 0.05) compared

with men in both protocols. During the ±occlusion period,

in the PECO protocol boys exhibited a greater bradycardic

response than men (P \ 0.05). During the same period, the

% change in HR in boys was not significantly different

between the PECO and non-PECO protocols, whereas in

men significant differences (P \ 0.05) between the PECO

than the non-PECO protocols were observed. During the

recovery period, no differences were observed between

protocols in both groups. In the PECO protocol, the %

change in HR in men was significantly lower in the

recovery period compared with the ?occlusion period,

whereas in boys no differences between the two periods

were observed. In the non-PECO protocol, % change in HR

was similar between the two periods (-occlusion and

recovery), within each group.

Although boys had lower absolute SV than men, the

percent change from baseline in SV was higher (P \ 0.05)

in boys than in men, in the respective periods, during the

course of both protocols (Fig. 4c). During HG, % change in

SV was not significantly different in the PECO compared

with the non-PECO protocol within each group. During the

±occlusion period, % change in SV in boys was not sig-

nificantly different in the PECO compared with the non-

PECO protocol, whereas % change in SV in men was

significantly lower in the PECO than in the non-PECO

protocol (P \ 0.05). During the recovery period, % change

in SV in boys was higher in PECO than in non-PECO

Fig. 1 Representative raw data from systolic and diastolic blood

pressure recordings during the Post-exercise occlusion (PECO)

protocol (a and b, in a child and in an adult, respectively) and the

control (non-PECO) protocol without post-exercise occlusion (c and

d, in a child and in an adult, respectively)

Eur J Appl Physiol (2013) 113:827–838 831

123

Fig. 2 Mean arterial blood pressure (a), heart rate (b), stroke volume

(c), and total peripheral resistance (d) data expressed in absolute

values (mean ± SE), in pre-adolescent boys and men. PECO post-

exercise occlusion protocol, non-PECO control protocol without post-

exercise occlusion. Significantly different at *P \ 0.05 versus the

non-PECO protocol within the same group in the same time-period,

significantly different at �P \ 0.05 versus baseline within the same

protocol, in boys, significantly different at �P \ 0.05 versus baseline

within the same protocol, in men, significantly different at §P \ 0.05

versus men within the same protocol and testing period, significantly

different at #P \ 0.05 versus the previous testing period in the PECO

protocol. Significantly different at ^P \ 0.05 versus HG exercise

within the same protocol and group

Fig. 3 Representative raw data showing the heart rate decline and second degree decay fitting, during the first minute of recovery following

handgrip exercise in a child (a) and an adult (b)

832 Eur J Appl Physiol (2013) 113:827–838

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(P = 0.05), whereas no differences were observed in men.

During the same period (recovery), % change in SV has

higher (P \ 0.05) in boys than in men, in the respective

protocols.

Percent change in TPR is depicted in Fig. 4d. During

HG, % change in TPR was not significantly different

between boys and men, in the respective protocols. During

the ±occlusion period, % change in TPR was higher

(P \ 0.05) in the PECO protocol than the respective values

in the non-PECO protocol within each group; % increase in

TPR was lower (P \ 0.05) in boys than in men in the

PECO protocol, whereas in the non-PECO protocol the %

decrease in TPR was greater in boys than in men. During

recovery, a greater (P \ 0.05) decrease in TPR was

observed in boys than in men with no differences between

protocols.

Baroreceptor sensitivity

Absolute BRS data during the protocols in boys and men

are presented in Fig. 5a. The number of slopes per minute

ranged from 3.3 to 18.0 in boys (average 8.0 per minute)

and 3.0 to 27.0 in men (average 16.1 per minute). During

baseline, BRS values were not significantly different

between the two groups. During HG exercise, a significant

decline (P \ 0.05) from baseline in BRS was observed in

boys and in men. During this period, no significant dif-

ferences in BRS were detected between the two groups

within the PECO and non-PECO protocols. During the

±occlusion period, BRS values in boys were significantly

lower (P \ 0.05) than the respective values in men. BRS in

the ?occlusion was lower than -occlusion in boys and in

men (P = 0.05). During recovery, boys in the PECO

Fig. 4 Percent change from baseline in mean arterial pressure (a),

heart rate (b), stroke volume (c), and total peripheral resistance (d),

during handgrip (HG) exercise, ±occlusion, and recovery in boys and

men. PECO post-exercise occlusion protocol, non-PECO control

protocol without post-exercise occlusion. Significantly different at

*P \ 0.05, versus the non-PECO protocol in the same testing period,

within the same age group. Significantly different at �P \ 0.05,

versus HG within the same protocol, in boys. Significantly different at�P \ 0.05, versus HG within the same protocol, in men. Significantly

different at §P \ 0.05, versus men within the same protocol and

testing period. Significantly different at #P \ 0.05, versus ±occlusion

within the same protocol and group

Eur J Appl Physiol (2013) 113:827–838 833

123

protocol exhibited lower BRS values than men (P \ 0.05),

whereas no differences were observed between the groups

in the non-PECO protocol.

Heart rate variability

At rest, the LF/HF and the SD2/SD1 heart rate variability

indices were significantly lower in children compared

with adults (LF/HF 1.79 ± 0.26 vs. 2.99 ± 0.59 in boys

and in men; SD2/SD1 2.26 ± 0.15 vs. 3.08 ± 0.17

in boys and in men). During HG exercise, the SD2/SD1

index significantly increased in men and was higher

(P \ 0.05) than the respective value in children (boys

3.2 ± 0.3 vs. men 5.1 ± 0.7), whereas no significant

differences were observed in the immediate post-exercise

period between boys and men (2.1 ± 0.1 vs. 2.5 ± 0.2,

respectively).

Next, the RMSSD index of HRV was assessed, as an

index of parasympathetic activity (Fig. 5b). During base-

line, no significant differences were observed in RMSSD

values between groups. During HG, RMSSD decreased

(P \ 0.05) from baseline values, in both groups. However,

RMSSD was not significantly different in the PECO

compared with the non-PECO protocol, within each group.

During the ±occlusion period, RMSSD in boys was not

significantly different in the PECO compared with the

non-PECO protocol, whereas RMSSD in men was lower

in the PECO than the non-PECO protocol. During this

period, RMSSD values in boys were similar to baseline

values, whereas in men they were higher (P \ 0.05) than

baseline.

Discussion

The major finding of this study was that in healthy lean

boys compared with men, the metaboreflex elicited an

increase in AP similar to that evoked in adults; however,

the interplay among the mechanisms that controlled MAP

during the metaboreflex were different in children than in

adults. During the activation of the muscle metaboreflex,

we observed (1) a lower increase in peripheral resistance

and a greater bradycardia (relative to baseline) in children

compared with men, (2) a fast restoration of vagal activity

to the non-occlusion levels in the young age group, and (3)

a significant attenuation in BRS in both age groups. During

post-exercise recovery, in addition to the faster rate of HR

decay and vagal reactivation, boys exhibited a greater

decrease in TPR than men.

Hemodynamic and autonomic responses during rest,

exercise, and recovery

Boys displayed lower MAP and higher HR (in absolute

terms) than adults, at rest and during exercise, confirming

previous findings (Turley 2005; Goulopoulou et al. 2010).

In addition, boys exhibited differences in resting and

exercise measures of sympathovagal interaction compared

with adults. A possible interdependency of cycle length and

time domain indices (such as SDNN, and pNN50) has been

previously suggested (Zaza and Lombardi 2001). However,

changes in time domain HRV indices also occur indepen-

dently of heart rate, as shown in advanced stages of heart

failure, where an extreme depression of HRV may be

Fig. 5 a Baroreceptor Sensitivity and b root mean square of

successive differences (RMSSD), during baseline, handgrip (HG)

exercise, ±occlusion, and recovery in boys and men. PECO post-

exercise occlusion, non-PECO control protocol without post-exercise

occlusion. Significantly different at *P \ 0.05, versus the non-PECO

protocol within the same testing period and group. Significantly

different at �P \ 0.05, versus baseline within the same protocol, in

boys. Significantly different at �P \ 0.05, versus baseline within the

same protocol, in men. Significantly different at §P \ 0.05, vs. men

within the same protocol and testing period

834 Eur J Appl Physiol (2013) 113:827–838

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observed that is too large to be justified by heart rate levels

(Lucini et al. 2002; Zaza and Lombardi 2001; Task Force

of the European Society of Cardiology and the North

American Society of Pacing and Electrophysiology 1996;

Malliani et al. 1994). In support of this view, the differ-

ences in the magnitude of increase in SD2/SD1 during

exercise between children and adults in this study are too

large to be accounted only by the differences in HR. In

addition, the LF/HF and the Poincare plot indices that we

used for assessing sympathovagal modulation are consid-

ered to be devoid of intrinsic rate dependency (Zaza and

Lombardi 2001; Agelink et al. 2001; Pagani et al. 1993).

Therefore, although intrinsic differences in heart rate and

blood pressure existed between children and adults, our

findings in HRV parameters cannot be attributed to basal

HR differences. Furthermore, to account for the differences

in basal hemodynamic variables between the two groups

we also present and discuss the relative changes from

baseline levels. During HG, the change (%) in HR was

lower in children than in adults suggesting possibly a heart

rate dependency with the muscle mass involved (Lewis

et al. 1983; Seals 1993). The lower HR increase (in relative

terms) in children than in adults, was mirrored by a lower

SD2/SD1 index, suggesting a different sympathovagal

balance during exercise between groups. During exercise, a

parasympathetic withdrawal was evident in both groups. In

adults, HR was the key contributor to the MAP elevation

during exercise, with no changes in SV. In children,

however, the lower increase in exercise HR was counter-

balanced by an increase in SV, resulting in similar MAP

levels to those observed in adults.

The rate of HR decay during the first minute of recovery

is considered a marker of cardiac parasympathetic outflow

(Buchheit et al. 2007; Imai et al. 1994). Two components

are involved in this immediate post-exercise heart rate

recovery, an initial rapid decrease and a subsequent slow

decrease (Imai et al. 1994). Imai et al. (1994) reported that

the initial rapid component depends on vagal reactivation,

since it disappeared with atropine or atropine plus pro-

pranolol administration and was independent of the exer-

cise intensity. The second component was prolonged by

atropine and this prolongation was significantly attenuated

by administration of propranolol suggesting that this

component depends on sympathetic along with parasym-

pathetic reactivation. In accordance with studies using

dynamic exercise (Hebestreit et al. 1993; Ohuchi et al.

2000; Zafeiridis et al. 2005; Dipla et al. 2009), we also

report a faster post-exercise HR recovery in children. In

some studies, non-autonomic factors were suggested to be

involved in the fast HR recovery in children, such as a

faster clearance rate of metabolites (Falk and Dotan 2006;

Ratel et al. 2006). However, the lower time constants

during the first minute of recovery in children compared

with their adult peers indicate, at least partly, that in HR

recovery the vagal involvement is maturity dependent.

During post-exercise recovery, children displayed a

greater decrease in TPR compared with adults and a res-

toration of RMSSD and BRS to baseline, whereas an

overshooting in RMSSD and BRS was observed in adults.

It should be noted, however, that vagal-related HRV

indices mirror the magnitude of modulation in parasym-

pathetic outflow and not the overall parasympathetic tone

per se (Buchheit et al. 2007). During the same period (i.e.

post-exercise recovery), a rise in SV (relative to baseline

levels) was observed in both groups. This post-exercise

rise could be could be attributed to several parameters,

such as an overshoot in cardiac function due to a transient

mismatch between cardiac contractility and the diminish-

ing vascular resistance and the rapidly induced bradycar-

dia (i.e. through longer filling time and the involvement of

the Frank-Starling mechanism) (Nottin et al. 2002; Shoe-

maker et al. 2007) and/or a change in peripheral demands,

central blood volume mobilization, and hence a change in

venous return (Shoemaker et al. 2007). In this study, the

greater bradycardia and the greater decrease in TPR at the

cessation of exercise in children possibly contributed to

the greater magnitude of increase in SV in children

compared with adults; however, differences between

children and adults in ventricular filling pressure and

cardiac afterload, central blood volume mobilization,

vascular peripheral resistance, and heart size should also

be taken into consideration.

Metaboreflex evaluation and mechanisms involved

To our knowledge, this is the first study that compared the

mechanisms involved in the MAP control during the me-

taboreflex in children and adults. Following isometric

exercise, a period of circulatory occlusion was used for

maintaining an activated metaboreflex. This technique

traps metabolites in the previously exercised muscle and

allows the isolation of the metaboreflex, while central

command and mechanoreflex are no longer active due to

the cessation of exercise (O’Leary 1993; Iellamo et al.

1999). Post-exercise occlusion resulted in similar MAP

elevations in children and adults. The comparable metab-

oreflex-induced MAP response could indicate a higher

sensitivity of group IV afferents in children, as lower lac-

tate concentration has been consistently reported in chil-

dren versus adults during strength activities performed at

similar relative intensities (Zafeiridis et al. 2005; Ratel

et al. 2006). The acid-sensing ion channel (ASIC) has been

recently shown to play a role in evoking the metaboreflex

in response to lactic acid (Hayes et al. 2008). Whether its

function is different in children from adults requires further

investigation.

Eur J Appl Physiol (2013) 113:827–838 835

123

In both groups, the metaboreflex MAP elevation was

evoked by a prominent increase in TPR, indicating an

augmented sympathetic tone (Kaufman et al. 1983;

Mitchell et al. 1983). This increase in TPR was greater in

men than in boys. The marked reduction in HR during this

period, in both groups, also supports a restoration of vagal

activity (Nishiyasu et al. 1994; Iellamo et al. 1999).

However, boys demonstrated a greater bradycardia (rela-

tive to their baseline levels) during the metaboreflex than

men and no further decreases in HR were observed in the

post-occlusion period, whereas in men, a further brady-

cardic response was observed in the post-occlusion period.

In children, the similarities in RMSSD values during

occlusion and non-occlusion also indicate a complete

reactivation of parasympathetic activity. However, in adult

males, the lower RMSSD in occlusion compared with non-

occlusion indicates an incomplete sympathovagal restora-

tion to baseline during this period. In support of our results,

previous studies showed that individuals with greater

enhancement of cardiac parasympathetic tone had a greater

bradycardic response to post-exercise occlusion (Watanabe

et al. 2010). However, the possibility that higher plasma

norepinephrine concentration during exercise in adults than

in children attenuated the parasympathetic control of HR

(Miyamoto et al. 2003) and exerted the incomplete resto-

ration of vagal tone during the metaboreflex cannot be

excluded. Differences in fiber type composition (higher

percentage of type I fibers in children), as well as in the

glycolytic capacity and mitochondrial content between

children and adults could also be potential mechanisms

influencing the sympathovagal drive during the metabore-

flex (Saito 1995; Seals 1993).

The return of heart rate to baseline (approximately

?1 bpm) during the metaboreflex observed in this study is

in agreement with previous reports in adult humans and

animals (Crisafulli et al. 2003; Iellamo et al. 1999; Nishi-

yasu et al. 1994). As previously shown by O’Leary (1993),

HR declines although BP remains elevated, due to the

overwhelming effect of cardiac parasympathetic activation.

The slightly higher HR in occlusion compared with non-

occlusion (by 5 bpm) observed in our adult participants is

possibly attributed to a predominance of cardiac sympa-

thetic activity over vagal activity exerted during the

metaboreflex. Fisher et al. (2010) showed that this minor HR

differentiation is abolished by b-adrenergic blockade and is

greater following higher intensity exercise. The magnitude

of this HR response could also be affected by physical

fitness levels, aging, or disease states (Notarius et al. 2011).

Notarius and Floras (2001) suggested that although the

involvement of HR during the metaboreflex is minimal or

negligible in healthy subjects, a higher contribution of HR

can be observed in disease states, as in heart failure, when

the diminished vagal drive may be insufficient to counter

the augmented sympathetic drive. Therefore, our HR data

in young physically active men with relatively high levels

of MVC, do not necessarily apply to older, sedentary

individuals or in disease states. If individuals with a

diminished vagal drive were tested (such as sedentary

adults or older individuals), the differences in the HR

decline during the metaboreflex compared with children

could have been greater. Moreover, since obesity levels can

influence the metaboreflex control of MAP and sympath-

ovagal drive in children (Dipla et al. 2010) and in adults

(Negrao et al. 2001) our data apply to non-obese

individuals.

Baroreceptor sensitivity

Our findings showed that BRS was attenuated during

exercise, confirming previous reports in adult humans

(Iellamo et al. 1997), and in animals (Burger et al. 1998),

using the sequential method of BRS analysis (Sala-Mer-

cado et al. 2007). This technique allows the measurement

of baroreceptor sensitivity around the operating point and

does not indicate the maximal or total sensitivity. Thus,

although our result does not provide information on the

whole range of the stimulus response curve during exer-

cise, it allows a non-invasive measurement of baroreflex

gain during exercise (Iellamo et al. 1997). To our knowl-

edge, no previous studies compared the muscle metabore-

flex-baroreflex interaction between children and adults.

Although BRS contributed to the metaboreflex, possibly by

lessening its opposition to the MAP elevation, the degree of

involvement was similar between the two groups. Sala-

Mercado et al. (2007) also demonstrated a reduction in

BRS during metaboreflex in conscious dogs. Termination

of the occlusion restored BRS to the non-occlusion levels.

Turley (2005) has previously shown that in the post-

occlusion period (following pressure release in the cuff),

MAP remained higher in children than in adults. The

authors speculated that this phenomenon was due to an

activation of a more sensitive arterial chemoreflex in chil-

dren; however, the determinants of that MAP response were

not examined. In this study, a prominent MAP was observed

in boys during the post-occlusion period (recovery period in

the PECO protocol), induced primarily by increased SV,

while bradycardia and low TPR levels were evident. A more

sensitive arterial chemoreflex or increased muscle blood

flow and faster washout rates of metabolites from the tissue

in the cessation of occlusion in children could be possible

mechanisms that require further examination.

Limitations

The present study is limited by its reliance on indirect

measures of the autonomic nervous system and estimation

836 Eur J Appl Physiol (2013) 113:827–838

123

of stroke volume. However, the HRV and sequence BRS

analysis are valuable tools widely used in clinical practice

and physiological research that provide an evaluation of

autonomic modulation (Lucini et al. 2002). These tech-

niques have the merit of being non-invasive and hence are

well suited for young individuals and healthy children.

Moreover, only indirect inferences can be drawn from the

present data on the underlying mechanisms of our obser-

vations (i.e. the higher sensitivity of group IV fibers,

maturity, lower lactate production, norepinephrine levels,

fiber composition, chemoreflex differences, etc. in children

than adults) and additional factors, independent of the

autonomic state, should not be excluded.

In conclusion, in pre-adolescent boys the cardiovascular

system exhibited a faster ability to re-establish flow in

response to stressor mechanisms (i.e. exercise) than in

adults. During submaximal isometric exercise of similar

relative intensity, boys exhibited an attenuated heart rate

response compared with men, linked at least partly to

different sympathovagal balance. During the isolated me-

taboreflex, boys were capable of eliciting a similar MAP

rise to that elicited by men; however, the interplay of

mechanisms controlling this rise was different between the

two age groups. During the metaboreflex, maturation did

not exert differential effects on the BRS response; how-

ever, in children the rapid vagal outflow persisted, differ-

entiating the TPR and the heart rate responses compared

with adults.

Conflict of interest All the authors declare no conflict of interest.

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