Reports from cross-sectional studies suggest that endurance-trained subjects have a higher parasympathetic activity than untrained subjects (10,14,18,19). Intensive endurance training has been shown to have an effect upon autonomic regulation promoting vagal predominance (15), and it has been associated with the well-known resting bradycardia of endurance athletes (10). However, these studies are based on measurements of parasympathetic activity in different target organs. Whether the parasympathetic activity measured in one target organ reflects a general parasympathetic function or if it is only applicable for those specific nervous pathways involved remains to be clarified.
The heart is widely used as a target organ of parasympathetic activity, primarily by measurements of the variations of time intervals between beats calculated from R-wave to R-wave (RR) from an ECG, defined as HR variability (HRV) (29). Generally, HRV assessments are based on time-domain or frequency-domain analysis of direct RR intervals or the difference between successive RR intervals. Another assessment method previously used on athletes is pupillometry (14), where the pupil reaction to a light stimulus reflects autonomic function. Pupillometric and HRV parameters were shown to significantly correlate during exercise (18), suggesting that pupillometry could be used as a complementary tool in the evaluation of cardiac autonomic function. However, the agreement between these methods targeting different organs has not been assessed previously.
Pichon et al. (25) demonstrated that subjects with increased bronchial hyperresponsiveness (BHR) had higher vagal tone. The autonomic nervous system mediates the contraction and relaxation of bronchial smooth muscle with cholinergic parasympathetic nerves stimulating bronchoconstriction, whereas sympathetic nerves bronchodilate (7,9). Therefore, increased parasympathetic activity could also predispose to increased bronchomotor tone and further to susceptibility to bronchospasm (23). Increased risk for asthma has been recognized in elite endurance athletes (6), and recently, Couto et al. (11) demonstrated a relationship between parasympathetic activity and BHR in asthmatic swimmers. In addition, it has been suggested that the protective effect of ipratropium bromide, an inhaled anticholinergic drug blocking efferent cholinergic pathways in the airways, on exercise-induced asthma may be related to cardiac vagal activity, by evaluating the HRV response to a 4-s exercise test (4sET) (1), in children (19) and cross-country runners (18). Furthermore, there is an inverse correlation between the bronchodilating effect of inhaled ipratropium bromide (iIB), but not inhaled salbutamol, a general bronchodilating agent, and the cholinergic sensitivity measured by a methacholine provocation challenge in elite cross-country skiers (28). These results suggest that BHR in endurance athletes is related to increased parasympathetic (bronchial) activity and that athletes with asthma respond better to anticholinergic agents than β2-agonists.
To our knowledge, the repeatability of parasympathetic activity measurements in different target organs has not previously been compared. In addition, the interchangeability of methods measuring parasympathetic activity in different target organs is not clear, despite reports of a general increased parasympathetic activity of endurance athletes in the literature. Therefore, the main objectives of the present study were to assess the repeatability of two parasympathetic activity measurement procedures, pupillometry and 4sET, and to assess the agreement between them. The secondary aim was to identify a possible relationship of both procedures to the effect of ipratropium bromide inhalation on pulmonary function. Finally, we wanted to assess interrater variability of 4sET.
MATERIALS AND METHODS
Subjects and study design
Forty subjects were enrolled in a cross-sectional open study that was carried out at the Norwegian School of Sport Sciences (NSSS), Oslo, Norway. The subjects (14 females and 26 males) were aged 16–40 yr and consisted of competitive swimmers from regional clubs, elite cross-country skiers, and students from the NSSS. Subject characteristics are given in Table 1. To be included in the study, they had to be free from any respiratory disease 2 wk before testing. They were asked not to drink coffee or smoke 2 h before testing and not to exercise on the testing day. Exclusion criteria were the use of any systemic medication with CNS effects, any topical eye treatment, systemic conditions with known ocular involvement, or any other diseases that would influence the results or limit the patient’s ability to carry out the tests. Included subjects were asked to fill out a questionnaire before starting the procedures, to assess eligibility. The questionnaire also included information about sport practice characteristics, medical diagnoses of asthma, allergic diseases, or other medical conditions, as well as any current medication and smoking status. All participants signed an informed consent. The study was carried out in accordance with the Declaration of Helsinki and was reviewed by the Regional Committee for Medical and Health Research Ethics.
Data collection was performed in one visit, during mornings between 8:00 and 12:00 a.m. by standardized procedures. Either subjects attended the laboratory at NSSS (n = 33) or testing was performed at the athlete training facility of the Sogn Swimming Pool, Oslo, Norway (n = 7). Height and weight were measured. Baseline HR was measured with Polar Electro® (RS-800CX/G3; Oy, Kempele, Finland) HR monitor after 15-min resting in a semidark room. HR during the 15-min rest period ranged between 32 and 77 bpm, indicating low to none sympathetic influence. Pupillometry was then performed, followed by a 4sET. After 5 min, both pupillometry and 4sET were repeated. Finally, a reversibility test was performed.
The subjects spent 15 min in a semidark and quiet room to allow their eyes to adjust to the low lighting levels before measurement. Pupillary measurements were taken with the portable infrared PLR-200™ pupillometer (NeurOptics Inc., CA). The subjects were instructed to focus with the eye that was not being tested on a small object 3 m away. The PLR-200™ pupillometer stimulates the eye with a light flash of 180-nm peak wave light and then captures and analyzes a rapid sequence of digital images of the pupil. Each recording lasted for about 3 s. One pupil light response curve to each eye was recorded for each subject, starting with the left eye. In case of blinking, measurement was repeated. The pupil radius is controlled by both parasympathetic and sympathetic nervous systems (20). The pupil constriction response to a light stimulus represents parasympathetic activity, and the dilatation face represents sympathetic activity. The following parasympathetic parameters were recorded for analysis in the present study: the percent of pupil constriction (CON), the average and the maximum constriction velocities (ACV and MCV, respectively), and constriction amplitude (AMP) calculated by subtracting the minimal diameter from the maximal diameter. The mean values of the left and right eyes were calculated and used for further analyses, as previously reported (11). Among the 40 included subjects, two subjects used contact lenses during pupillometry.
The 4sET consists of unloaded pedaling as fast as possible on a cycle ergometer from the fourth to the eighth second of a 12-s maximal inspiratory apnea (1). The subject remains seated on the cycle ergometer. After the HR stabilization at rest, four verbal commands were given to guide the actions to be sequentially performed at 4-s intervals, given in the following sequence: 1) a fast maximal inspiration, primarily through the mouth; 2) as fast as possible cycling; 3) sudden stop cycling; and 4) expiration.
To quantify the cardiac vagal modulation, the ratio of the RR interval immediately before starting the exercise and the shortest RR interval during the exercise were used to calculate the cardiac vagal index (CVI), according to Araujo et al. (2). A high index indicates higher parasympathetic activity. Two 4sET maneuvers were performed, allowing the HR to return to its baseline values before the next measurement, and the highest CVI was used for analysis (3). The CVI was calculated by two experienced raters to assess interobserver agreement.
Ipratropium bromide (0.500 mg·mL−1) was mixed in 1 mL of NaCl and delivered through a Sidestream nebulizer (Respironics Respiratory Ltd, Chichester, UK) connected to a CR60 compressor (Medicaid Ltd, West Sussex, UK) at a flow rate of >6 L·min−1. Lung function by maximal expiratory flow volume loops was measured using a MasterScreen Pneumo spirometer (Jaeger GmbH, Würzburg, Germany), before and 45 min after inhalation (22). The following variables were recorded: forced vital capacity, forced expiratory volume in 1 s (FEV1), and forced expiratory flow at 50% of vital capacity (FEF50), and the predicted values were according to Quanjer et al. (26). Clinical significant reversibility was defined as a ≥12% and 200-mL increase in FEV1 after inhalation (26). Antiasthmatic medication was withheld before testing. Inhaled short acting β2-agonists were withheld for 8 h before testing; inhaled long-acting β2-agonists, theophylline, and leukotriene antagonists were withheld for the last 72 h; anticholinergics were withheld for the last 12 h; antihistamines were withheld for the last 7 d; and orally administered glucocorticosteroids were withheld for the last month. Inhaled corticosteroids were not to be used on the day of testing.
Demographic data and results are given as mean values with SD, or, in case of categorical data, as counts (%). Correlations were assessed by Spearman’s correlation coefficients. Differences between two means were analyzed by Student’s unpaired t-tests after tests for normality. Differences in categorical data were assessed by χ2 or Fisher’s exact tests. Associations between the two methods were assessed by linear regression analysis with CVI as the outcome variable. Independent variables were CON (%), AMP (mm), ACV (mm·s−1), and MCV (mm·s−1). Confounding variables were age, sex, height, weight, BMI, number of training sessions per week, and time of test. Agreements between the two methods as well as between two CVI raters were assessed with intraclass correlation (ICC) (two-way mixed, absolute agreement). P values < 0.05 (5%) were considered statistically significant. To assess the repeatability of each method, Bland–Altman plots were used. A sample size of 40 achieves 81% power to detect a difference of 0.30 between the null hypothesis low-grade correlation and the alternative hypothesis high-grade correlation of 0.70 using a two-sided hypothesis test with a significance level of 0.05. Statistical analyses were performed with Statistical Package of Social Sciences (SPSS) version 20.0 and MedCalc® Statistical Software version 12.
Repeatability for pupillometry
No differences were observed between the means of the first and second pupillometry trials in either of the pupil constriction variables. Bland–Altman plots (Fig. 1A–D) show no signs of a trend, and the variability is consistent across the graphs. The means of differences (with limits of agreement (LoA)) for the four pupillometry variables were CON = 1.21% (−3.59 to 6.02), AMP = 0.05 mm (−0.28 to 0.39), ACV = 0.21 mm·s−1 (−0.71 to 1.12), and MCV = 0.19 mm·s−1 (−0.63, 1.01). Correlations between trial 1 and trial 2 were 0.87 for CON, 0.83 for AMP, 0.64 for ACV, and 0.82 for MCV. All correlations were highly significant (P < 0.0001).
Repeatability for 4sET
No differences between the mean values of the two trials of the 4sET were found (P = 0.73). The Bland–Altman plot (Fig. 2) shows a mean difference value of 0.005 with LoA from −0.313 to 0.324. The wide LoA indicates a poorer repeatability as compared with the pupillometry test. The Bland–Altman graph shows no signs of a trend, and the variability is consistent across the graph.
Agreement between pupillometry and 4sET
A weak linear relationship was found between the two methods (Fig. 3A–D), and the results of the linear regression analysis show that there were no associations between the CVI and the pupillometry parameters (Table 2). Correlations between the pupillometry variables and CVI are given in Table 3. ICC coefficients were all weak and nonsignificant and varied around −0.01 (95% CI, −0.088 to 0.112) depending on the pupillometry variable. Absolute values of CVI from the 4sET are available in online supplements (see the Table, Supplemental Digital Content 1, absolute values of parasympathetic parameters, http://links.lww.com/MSS/A577).
The relation of both procedures with the effect of ipratropium bromide inhalation on pulmonary function
No significant correlations were observed between ΔFEV1 and the parasympathetic variables of pupillometry or CVI (Table 3). Mean change in FEV1 after inhalation of ipratropium bromide was 4.4% (2.5, 5.8) and 164.9 mL (99.9, 229.9). No difference was observed between competitive (n = 29) and noncompetitive athletes, nor asthmatic (n = 7) and nonasthmatic subjects. Two subjects had a positive reversibility test to ipratropium bromide. Both subjects were male competitive athletes, within swimming and cross-country skiing. Their FEV1 at baseline was 76% of predicted values and 89% of predicted values, respectively. The swimmer had doctor-diagnosed asthma and was treated with natural homeopathic products.
Interobserver determination of the CVI
A significant correlation between two different raters of CVI is shown by ICC ri = 0.91 (0.82, 0.95) (P < 0.001) for trial 1 and ri = 0.89 (0.80, 0.95) (P < 0.001) for trial 2. No differences between the mean values of the two trials were found (P = 0.73). Bland–Altman plots showed good agreement between the rates with a mean of differences of 0.03 with LoA from −0.14 to 0.19 [see Figure, Supplemental Digital Content 2, agreement between raters of the CVI, http://links.lww.com/MSS/A578].
The results from the present study showed good repeatability of both methods of parasympathetic activity measurement, pupillometry and 4sET, with a generally better repeatability for the pupillometry variables as compared with the CVI. In addition, the interrater agreement of interpreting the CVI proved to be consistent. On the other hand, we found no agreement between parasympathetic activity measured in the heart using a 4sET protocol and in the eye using pupillometry in athletes and noncompetitive active subjects. These findings suggest that parasympathetic activity measured in different target organs is dependable, although not similar between organs and may differ within the same subject. Kaltsatou et al. (18) found correlations between parasympathetic indices of HRV and pupillometric variables in athletes and sedentary subjects during maximal exercise, which are not in line with the results from the present study. However, during maximal exercise, the influence of the sympathetic branch of the autonomic function must be considered and the complexity of the sympathetic/parasympathetic interplay is challenging when measuring the activity of each branch separately. We found no interindividual agreement between parasympathetic activities measured in the pupil, the heart or the bronchi (by reversibility to an anticholinergic bronchodilator). The results from the present study suggest that the regulations by vagal activities measured in different organs are under the influence of largely independent mechanisms and are functionally unrelated, both at rest and at the onset of exercise. Thus, measurements of parasympathetic activity of the heart, the pupil, or the bronchi are not necessarily comparable.
In the present study, HRV was assessed during the first 4 s of exercise. The 4sET is shown to be a specific method for measuring parasympathetic influence upon the heart at the onset of exercise. The parasympathetic blockade by intravenous atropine abolished the HR increase during 4 s of cycling, whereas the β-adrenoceptor blockade by propranolol did not (2). This indicates that the parasympathetic withdrawal is dominant in the HR response in the first seconds of exercise and not the sympathetic activation. Pupillometry has the advantage of providing data of both branches of the autonomic nervous system separately, with the pupil constriction phase representing the parasympathetic tone and the pupil dilatation representing the sympathetic activity (5).
Kaltsatou et al. also found a greater parasympathetic activity in endurance athletes compared with power athletes and sedentary control subjects, which corresponds with previous reports (10,14). In the present study, we found no difference between competitive athletes and recreationally active subjects (see Supplemental Table 1, SDC 1, absolute values of parasympathetic parameters, http://links.lww.com/MSS/A577). However, these noncompetitive athletes were very active, which may explain the lack of statistical difference between the groups. In the present study, we observed no relationship between parasympathetic activity and the number of training sessions per week (data not presented). In addition, different assessment procedures and equipment used may explain part of the discrepancies between our results and those of Kaltsatou et al.
The lack of agreement found by ICC or linear regression analysis in the present study may be caused by the different parasympathetic pathways upon the target organs. Unlike the pupil, both the lungs and the heart are served by the vagus nerve (16). Thus, measurements of parasympathetic activity in the lungs and heart may be more comparable. A correlation between parasympathetic activity and bronchoconstriction as well as cold air inhalation has been reported (4,13,19). However, Horváth et al. (17) found no association between bronchial and cardiac vagal activity. This is in line with the results from the present study with the reversibility to iIB representing the inhibition of parasympathetic activity. However, seven of the subjects with asthma reported of taking inhaled corticosteroids, which may have influenced the results of the reversibility test.
Methacholine is a synthetic choline ester that acts as a nonselective muscarinic receptor agonist in the parasympathetic nervous system, whereas anticholinergic agents, such as ipratropium bromide, inhibit parasympathetic nerve impulses through competitive inhibition upon the muscarinic acetylcholine receptors in neurons and affect the involuntary movements of smooth muscles of the bronchi (12). Thus, both substances reflect opposite measures of bronchial parasympathetic tone and are shown to be closely associated in elite cross-country skiers (27). Also, it was previously shown that BHR to methacholine (PD20methacholine) correlates with parasympathetic parameters of pupillometry in asthmatic elite swimmers (11). Parasympathetic stimulation in the airways causes bronchoconstriction and is thought to contribute to the development of BHR and asthma in athletes of endurance sports (8).
Two male subjects reported current smoking, which could influence their respiratory status. Their lung functions were within normal values for FEV1 (93% and 103% of predicted values) and forced vital capacity (107% and 117% of predicted values), respectively. None had a significant improvement in FEV1 after inhaled ipratropium (>12%). Excluding these subjects from the analyses did not change the results.
In the present study, testing were performed at the same time of day, with the two noninvasive protocols carried out immediately following each other, and with the subjects seated in the same body position, on the ergometer bike. The two protocols used are both time efficient and easy to perform, which is a big advantage in both scientific and clinical practice. Additionally, both assessment methods have available mobile devices and therefore allows for testing performed in specific training or competitive environments. In the present study, HRV was measured using HR monitors that are shown to be a comparable option to ECG (28). The results of the present study support the work by Araújo et al. (2), which have demonstrated the reliability of 4sET. Intraday and interday variations of the CVI have previously been shown to be more reliable as compared with HR recovery after exercise (3). However, pupillometry variables showed a better reproducibility as compared with the 4sET.
The main parasympathetic outcome variables of pupillometry are CON (%), AMP (mm), and pupil constriction velocity (ACV or MCV (mm·s−1)), and the CVI is a ratio representing cardiac vagal activity. Because of different units of these outcome variables, it was inappropriate to assess the agreement between them using the Bland–Altman plot. In the analysis, we adjusted for confounding factors for HRV previously reported in the literature (28). However, the same confounding factors do not necessarily affect pupillometry. Filipe et al. (14) found that sex, age, height, weight, body mass index, or years of sports practice had no significant influence in the evaluated parameters of pupillometry. Interestingly, the same have been observed regarding the 4sET (3).
The subjects were asked to refrain from coffee 2 h before testing (11). However, 73% of the subjects (n = 30) did measurements early in the morning (8:00–10:00 a.m.) and reported they had not had coffee the same day (>8 h). The last 10 subjects were tested between 10:00 a.m. and 12:00 p.m., and five (two females and three males) stated that they had coffee between 7:00 and 8:00 a.m. that same day. They were all tested between 11:00 a.m. and 12:00 p.m. Although the tests were performed immediately following each other, intake of caffeine may have influenced the target organs differently and may thus have influenced the data. We compared the subjects who did not drink coffee with those who did and found no significant differences in parasympathetic variables of the pupil or heart (see Supplemental Table 1, SDC 1, absolute values of parasympathetic parameters, http://links.lww.com/MSS/A577). In addition, the five subjects had baseline resting HR (52.9 (10.75) bpm) similar to the subjects that did not drink coffee on the day of testing (53.8 (6.30), P = 0.070). However, this does not exclude the possible influence of caffeine, but it suggests that it may not have confounded our results.
The results from our study show no associations between parasympathetic activity measured in different target organs: the heart and the pupil. Additionally, no correlations were observed with reversibility to iIB reflecting bronchial vagal tone. These results suggest that parasympathetic activity measured in one organ is not transferable to another organ, and that these assessment methods cannot be used interchangeably.
The authors acknowledge the European Academy of Allergy and Clinical Immunology for the fellowship attributed to Mariana Couto, Professor Luis Delgado and Dr. Miguel Capão Filipe for their contribution in the critical discussion of the pupillometry protocol, Stian Roterud, M.Sc., for his contribution as the second rater of the cardiac vagal index, and all the subjects who kindly agreed to be involved in this project.
The authors have no conflicts of interest or financial ties to disclose. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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