Concurrent training effects on heart rate variability, blood pressure and fi tness of middle-aged men and women

Melissa Antunes * Valéria Bonganha ** Giovana Verginia de Souza ** Arthur Fernandes Gaspari ** Cleiton A. Libardi *** Mara Patrícia T. Chacon-Mikahil ** *Study and Research Group in Metabolism, Nutrition, and Exercise, State University of Londrina, Londrina, PR, Brazil. * * L a b o r a t o r y o f Exercise Physiology, School of Physical E d u c a t i o n , S t a t e U n i v e r s i t y o f Campinas, Campinas, SP, Brazil. * * * D e p a r t m e n t o f Physical Education, Center of Biological Sciences and Health, Federal Univers i ty of São Carlos, São Carlos, SP, Brazil. Abstract

Introduction related to level of fi tness [14][15][16][17][18] . Although a decline in autonomic control occurs in both genders, it can take place earlier in men due to the cardioprotective effect of estrogen 19 . Some studies indicate greater parasympathetic modulation and lower sympathetic modulation in women compared to men 20 . However another study 21 , have showed no signifi cant diff erences in autonomic modulation between genders. Th ere is evidence that these cardiovascular diff erences between men and women until middle age tend to disappear over the years 20,22 . To the best of our knowledge, just one study have shown that concurrent training may improve heart rate variability (HRV) and HR at rest, markers of autonomic control 23 . Interestingly, this study investigated only men who are known to have more cardiovascular benefi ts, as reduction in BP and improvements in autonomic control, arising from exercises training 8,23 . Th ese apparently sex diff erence in HRV response to concurrent training is reinforce for a second study conducted again by the Karavirta 24 group showing that 40 to 65 years old women did not improve autonomic control after similar concurrent training.
Th us, the eff ects of concurrent training in autonomic control markers of female, as well as the sex diff erence in training induce adaptation need to be investigated. Th e present study analyzes cardiac autonomic adaptations in response to 16 weeks of concurrent training and compares the results of male and female. Based on the above considerations, we hypothesizes that the training groups would show similar cardiorespiratory and neuromuscular adaptations, and further improvements in HRV parameters in both genders, with no diff erences between them, since the middle-age female volunteers of this study were postmenopausal women who have the same levels of autonomic control imbalance of the middle-age male volunteers 19,[25][26][27] .

Experimental design
Th e investigation was performed over a period of 16 weeks, this protocol was divided into two phases, each lasting eight consecutive weeks. The cardiorespiratory assessment, muscular strength test and rest cardiovascular evaluation were performed before and after the 16 weeks of experimental protocol keeping a minimum of 48 hours interval between teste and last training session. Th e training was performed three times a week and progressively supervised and sedentary group did not perform any type of physical exercise during this period.

Ethical aspects
After meetings with volunteer groups, those with the necessary prerequisites completed an initial interview and signed the Informed Consent approved by the Ethics Committee of the local University (CEP no. 250 and 251/2003, with addendums in 2007).

Participants
No sample size calculation was performed in this study. Th irty-two volunteers aged 40-60 years were included in the study, classifi ed as non-active according to the Baecke questionnaire 28 , and IPAQ 29 . The exclusion criteria adopted were: any complication that could be classifi ed as a risk factor for proposed program, detected in the clinical evaluation, biochemistry and/or during exercise; use of any medications that interfere in physiological responses to testing; non-availability of the volunteer to participate in the experimental procedures and/or training sessions.
For the female group there were additional criteria, such as a minimum absence of menstruation for 12-months, being postmenopausal and not using any type of hormone replacement therapy. Only volunteers who participated in at least 85% of the training sessions and were not absent for more than two consecutive training sessions were included in the fi nal sample. Th e groups were separated by gender into the following groups: male concurrent training (MCT), female concurrent training (FCT), male sedentary control (MSC) and female sedentary control (FSC) groups. TABLE 1 presents the general characteristics of the groups.
Th e volunteers who took part in the sedentary group were instructed not to practice any type of systematic physical activity during the period of the study. FIGURE 1 shows the fl ow of study participants.

Experimental protocols
Prior to the evaluation protocols, familiarization with the testing equipment was performed. Both the initial evaluations and re-evaluations were applied in the same period of the day to avoid any infl uence of circadian variations. All evaluations were performed at an ambient temperature of 22°C.
Clinical exercise testing was performed initially in order to diagnose and rule out the possibility of the occurrence of cardiac events during the later maximal exercise test and the proposed physical training. A minimum of 48 hours after the clinical examination, an evaluation of total body mass (mechanical scale, Filizola, Brazil) and height (wooden stadiometer) were performed according to the procedures described by Gordon, Chumlea and Roche 30 .

Blood pressure assessment
Blood pressure assessments were done after approximately 10 minutes of rest using a mercury sphygmomanometer and stethoscope. Th e participants were positioned supine and the same professional made the measurements. All measurements were taken in duplicate and the mean of two assessments was used. Th e formula for mean blood pressure (MBP) assessment was: where DBP is diastolic blood pressure and SBP is systolic blood pressure.

Cardiovascular assessment at rest
A minimum of 72 hours after the assessment of muscle strength, evaluation of HR was performed for the analysis of heart rate variability at rest, using a heart rate monitor (S810i -Polar® -Finland) to obtain the beat-to-beat records corresponding to the intervals between consecutive R waves of the electrocardiogram (iRR) 31,32 . Th e volunteers were requested not to talk and to stay awake during the protocol. After fi ve minutes of rest in the supine position, the recording was initiated and continued for a total of 30 minutes while the participant breathed spontaneously. Both the blood pressure and resting HR were measured immediately prior to the iRR collection to ensure the standard conditions of supine rest. Analysis of HRV in the time and frequency domains was performed through tachograms of iRR.
The first 10 minutes were discarded from the analysis and the most stable 256 consecutive points from the central region of the time series signal were selected by visual selection 33 . HRV Analysis® software, version 2.0 (Finland, 2008), was used to analyze the steady state and obtain average values of iRR. Th e variables pNN50 (percentage of adjacent RR intervals with 50-millisecond difference), RMSSD (square root of the average of the squared diff erences between adjacent normal RR intervals) and the Poincaré plot, obtained from the SD1 and SD2 33 in time domain and the LF (Hz) -low-frequency component HF (Hz) -high-frequency component, and LF / HF (ratio low components and high frequency) values in the frequency domain.

Cardiorespiratory Assessment
The cardiorespiratory test was conducted following the descriptions used in Libardi et al. 34 . Cardiorespiratory evaluation was then performed via a test protocol on a treadmill (Quinton TM55. Bothell, Washington, USA), with an initial speed of 4km/h for two minutes, followed by increases of 0.3km/h every 30s and a constant slope of 1% 34 , until physical exhaustion. Recovery was observed for a period of 4 minutes; the fi rst minute at 5 km/h, reducing 1 km/h every minute. Th roughout the stress test, gas exchange data were collected continuously, breath to breath, by means of a metabolic gas analysis system (CPX Medical Graphics, St. Paul, Minnesota, USA). Th e peak oxygen consumption (VO 2peak ) was defi ned as the average over a period of 30s during the fi nal stage of the incremental test, since none of the volunteers presented the criteria used to characterize maximal oxygen consumption35. Th is review was carried out in three stages: before training, after eight weeks of training, only to adjust the intensity of the training program, and at the end of the 16 weeks of physical training.

Muscle strength test
A minimum of 48 hours after the cardiorespiratory evaluation, muscle strength was determined through the one repetition maximum (1-RM) test on three exercises. Th e order of execution of the exercises was: Elbow extension in the supine position on a horizontal bench (bench press), knee fl exion and extension on a horizontal chair (leg press) and elbow fl exion (arm curl), with a fi ve minute interval between each set of exercises 36 . Prior to the start of the test protocol a familiarization protocol was performed in an attempt to reduce the eff ects of learning and establish reproducibility in the three exercises. All exercises were preceded by a warm-up series of 10 repetitions with approximately 50% of the load estimated by an experienced evaluator for the fi rst attempt at the 1-RM test. Testing began three minutes after the warm-up, during which the volunteers performed a single repetition with the expected load for 1-RM. Whether this was completed or not, a second attempt was given after an interval of three to fi ve minutes with a greater or lesser load (kg) than that previously employed. A third and fi nal attempt was made if the single maximum repetition load had not yet been determined. To determine the results of the1RM tests at baseline, we used the value of the highest load obtained after the test-retest.

Concurrent training protocol
Th e concurrent training protocol was composed of aerobic and resistance training performed in the same session. Th e training program consisted of three weekly sessions on alternate days (Monday, Wednesday and Friday), with an approximate duration of 60 minutes per session, for a period 16 consecutive weeks. Th e physical training protocol was guided and accompanied by Physical Education professionals and academics. Th is protocol was divided into two phases, each lasting for eight consecutive weeks. During the fi rst eight weeks of training, the participants initially performed resistance training that consisted of six exercises. Th ey perfonned three exercises for the lower body (leg press, leg extension, and leg curl) and three exercises for the upper body (bench press, lateral pulldown, and arm curl) with three sets of 10 repetitions and intervals of one minute; the session lasted approximately 30 minutes37, and the order of the exercises altered by segment. Next the participants performed walking and/or continuous running for 30 minutes on an outdoor athletic track, with intensity variation during the training session as follows: fi ve minutes below the ventilatory threshold (VT), 10 minutes at VT, 10 minutes above VT but below respiratory compensation point (RCP), fi ve minutes below VT 38 , totaling over 30 minutes of training. In the subsequent eight weeks, the resistance training session was held with the same series of exercises as in the previous weeks, with eight repetitions and intervals of 90 seconds37, also lasting about 30 minutes, however, the order of the exercises was according to joint. For aerobic training there was an adjustment in training zone intensity and length of time in each, fi ve minutes below the VT, 10 minutes above VT but below RCP, 10 minutes at RCP and fi ve minutes below VT, resulting in an additional 30 minutes of training. It is noteworthy that the total duration of the concurrent training session was about 60 minutes.
After the first eight weeks, the volunteers underwent cardiopulmonary reevaluation to adjust the intensity for the next step. Th e aerobic intensity of training related to the VT and RCP was monitored by means of test speed on the treadmill, since it was performed with a gradient of 1% in an attempt to reproduce the training conditions on the running track. Regarding the resistance training, load adjustment was performed weekly. Adjustments were made in the fi nal set of each exercise in the last week of the training session. Individuals were encouraged to perform the maximum number of repetitions and load was reset based on performance, using the following calculation: for each repetition exceeding the prescribed number of repetitions, the load was increased by 1 kg for lower limb exercises and 0.5kg for upper limb and trunk exercises. Th us, the loads used for training were consistent with the stipulated maximum repetitions for each exercise by following the principle of load progression 34 .

Statistical analysis
Data normality was verifi ed by the Shapiro-Wilk test. Non normal data were transformed by Ln (x) (pNN50) and log (X) (RMSSD, LFHF, SD1 and SD2) and then normality was confi rmed. A group X sex X time repeated measures ANOVA was used to compare all study variables. When a signifi cant F value was found, Bonferroni post hoc was performed to localize diff erences. To verify training eff ects on cardiovascular variables independent of sex a group (training N.16 and control N.16) X time (pre-and post-16 weeks) repeated measures ANOVA was used. Th e software package used for all analyses was STATISTICA 6.0 (StatSoft, Inc., Tulsa, OK, USA). Data are presented as means and standard deviations of non-transformed values and the level of statistical signifi cance was established at p≤0,05.
Mean ± standard deviation of the cardiovascular variables (HR, SBP and DBP) pre-and post-16 weeks of training or sedentary control by gender.   3 shows the hear rate variability (HRV) variables obtained pre-and post-16 weeks of training or sedentary control by gender. It was show that none of the components of HRV presented signifi cant group X sex X time interaction in ANOVA analyzes (all p > 0.05). ANOVA did not show group X sex X time interaction (all p > 0.05) for any of these variable. In the same way, two way ANOVA (group X time) did not show any interaction (all p > 0.05) for iRR (training: pre-894.  The TABLE 4 demonstrates the evaluation of cardiorespiratory values and muscle strength variables before and after the training period for the groups. Muscle strength signifi cant improvements was observed for the MCT in the leg press (p = 0.0001) and arm curl (0.0001) when compared to the sedentary groups. For FCT, a signifi cant increase was observed only in the bench press (p = 0.0130) and arm curl (0.0109) when compared to the sedentary groups. In the cardiorespiratory evaluation, signifi cant diff erences were observed for the MCT (p = 0.0001) and FCT (p = 0.0238). Th ere was no signifi cant diff erence in VO2peak from the baseline for the sedentary groups.
Mean ± standard deviation of VO2peak and 1 repetition maximum test (bench press, leg press and arm curl) before and after 16 weeks of training protocols.

Discussion
Th e main fi ndings of this study were that training protocol was not eff ective to alter resting HRV and BP, even though the VO 2peak improved for both training groups. In addition, both training groups have shown an increase in maximum upper body strength, but just men have increase in the lower limbs strength following concurrent training. Th us, these results contradict our hypothesis that 16 weeks of concurrent training would be able to promote similar gains in body strength and cardiac autonomic markers for both genders.
Some authors report that the association between aerobic and resistance training can lead to strength gains and cardiorespiratory fitness gains 24 , and improve the prevention of cardiovascular disease. However, there is little information regarding autonomic adaptations, particularly related to concurrent training 24 . Furthermore, the diff erent training methods used hinder comparisons between the results 39,40 . In this study, with regard to the variables related to muscular strength indicators in response to the proposed training protocol, the assumption of gains and no 'interference eff ect' for untrained subjects when concurrent training is prescribe was negated 41 . Although, studies have shown strength gains similar to those from a specifi c training protocol for the development of muscle strength 42,43 . However, few studies have investigated the eff ects of interference in the development of maximum strength in middle-aged individuals 40 .
Regarding blood pressure levels and HR, a recently published meta-analysis 8 , the results showed that combined training do not reduce SBP and MAP and just signifi cantly reduce DBP. In according, combined training proposed by us showed no reduction in any BP variable. One might imagine that the lack of eff ect of our training is due to the normal or little altered BP levels of our individuals. However, have been shown that in individuals with normal blood pressure or prehypertensive training also eff ects positively SBP and DBP, demonstrating the power of training as adjunctive therapy for high BP prevention in these populations 8 . In the present study a decrease in these BP values was not observed, although comparisons with literature are diffi cult due to the diff erent loads, intensities and type of training used, other studies should comparer the eff ects of concurrent training in BP values of normal BP and hypertensive subjects.
Studies on concurrent training and HRV are scarce. Karavirta et al. 12 showed improvements in cardiac dynamics in response to concurrent training (resting HR and HRV indicators) over 21 weeks of training in older men. In addition, signifi cant changes were observed only for the concurrent training when compared to other training protocols used (aerobic and with weights). There was a negative correlation between the reduction in HR at rest and increased HF (r = -0.81, p <0.001), which indicated greater vagal infl uence on cardiac autonomic function in the group undergoing concurrent training. Although in this study thus it could be speculated that the larger training volume was responsible for the best results obtained by the concurrent training group. In addition, another study 24 from the same group showed that the same protocol did not confer benefi ts on HRV scores in women.
Th e study of Verheyden et al. 44 evaluated the eff ects of a concurrent exercise training program on cardiac autonomic control in 14 sedentary men with a mean age of 62 ± 6.1 years. The training consisted of approximately 75 minutes of aerobic activity at 65-80% of heart rate reserve plus two sets of 20-30 RM (repetition maximum) with a moderate load. Th e results of this study demonstrated no significant changes in HRV parameters at rest, and did not provide evidence of a signifi cant increase in vagal modulation at rest after a year. Th ese results suggest that the manipulation of these training variables may be of great importance for obtaining improvements in cardiac control in this population. However, more research is needed to clarify this information with regard to equalizing training volumes.
According to the Dutra et al. 20 study, cardiac modulation diff ers between the genres, showing greater infl uence of the vagal autonomic component in women and the sympathetic component in men. Our results show no diff erence between the genres, neither in the baseline nor after the training protocol. Although a decline in earlier autonomic control in men may occur early because of the cardioprotective eff ect of estrogen 19 , in our study these diff erences were not found and one possible explanation is that women are postmenopausal. With regard to gender dependency, in the present study no diff erences in HRV variables were observed for any of the periods studied, corroborating data from another study 21 which reported no diff erences in cardiovascular autonomic responses, HRV variables.
Despite considering only the improvements expected from specifi c programs of physical training for the variables related to the cardiovascular and respiratory systems, it is noteworthy that the evidence from the present study suggests that the systematic practice of physical exercise produces protective effects against the development of chronic degenerative diseases in advance stages of life, probable helping not only providing an increase in life expectancy, but above all improving the health status of an individual, making exercise a crucial public health strategy.
Th e changes induced by training in the modulation of HRV were not observed in the present study. However, Karavirta et al. 24 study suggests that it may be possible to detect more subtle changes when measured during exercise, but not when measured at rest. Th is may have been a limitation of this study since the proposed training may be consideration of moderate-high intensity. Another limitation was the lack of homonal dosage in women, since at that age, it is suggested that women have lost considered levels of the hormone estrogen, which has cardioprotective eff ect in women.
Based on the results of the present study, it was concluded that the training protocol proved to be a good proposal for improving cardiorespiratory capacity and muscle strength particularly in mem, however without causing alterations in the analyzed cardiac autonomic indicators and BP. Th ese fi nds suggest a possible 'interference eff ect' on cardiovascular variables, but we can not prove this assumption without comparison with the isolated aerobic training protocol. Further studies should compare the eff ects of aerobis and concurrent trainng protocols in middle-age men and women upon cardiovascular variables. In addition the manipulation of training volume may increase concurrent training eff ects, this hypothesis can also be test by other studies.
The changes induced by training in the modulation of HRV were not observed in the present study. However, Karavirta 24 study suggests that it may be possible to detect more subtle changes when measured during exercise, but not when measured at rest. This may have been a limitation of this study since the proposed training may be consideration of moderate-high intensity. Another limitation was the lack of homonal dosage in women, since at that age, it is suggested that women have lost considered levels of the hormone estrogen, which has cardioprotective effect in women.