Effects of Ultratrail Running on Neuromuscular Function, Muscle Damage and Hydration Status. Differences According to Training Level

s of trail running races has exponentially grown in recent years. The present study aimed to: (a) evaluate the acute response of ultratrail racing in terms of neuromuscular function, muscle damage and hydration status; (b) analyze if responses could differ according to training levels. Twenty runners participated in the present study. The participants were divided into amateur training level (n= 10; 43.30 4.52 years) or high level competitors (n= 10; 41.40 6.18). Neuromuscular response (squat jump, countermovement jump and Abalakov jump), muscle damage (alanine aminotransferase, bilirubin, creatine kinase and leukocytes) and hydration status (sodium and creatinine) were evaluated before and after the Guara Somontano Ultratrail Race (108 km distance, with an accumulated

participants were divided into amateur training level (n= 10; 43.30 4.52 years) or high level competitors (n= 10; 41.40 6.18). Neuromuscular response (squat jump, countermovement jump and Abalakov jump), muscle damage (alanine aminotransferase, bilirubin, creatine kinase and leukocytes) and hydration status (sodium and creatinine) were evaluated before and after the Guara Somontano Ultratrail Race (108 km distance, with an accumulated slope of 5800 m). The height and power achieved by vertical jumps were lower after the race (p< 0.001). The post-race muscle damage and creatinine parameters increased in both groups (p< 0.001). The high-level group obtained lower percentages of change in squat jump and countermovement jump than the amateur-level group (p< 0.05). However, the increase in creatinine was greater for the high-level group (p< 0.05). Ultratrail racing reduces neuromuscular function and increases muscle damage. High-level runners showed less neuromuscular fatigue compared to amateur ones. Keywords:trail running; jump; neuromuscular; hydration 1. Introduction Ultratrail is a form of very long-distance mountain racing included in trail running. Given its extreme characteristics, this kind of racing can be considered very taxing, or even dangerous, for our organism [1]. Ultratrail racing is performed on routes with adverse orography. Its distance exceeds that of marathons [2,3], and ranges between 80 km and 300 km [4]. The fact that ultratrail runners face unstable environmental and weather conditions for long periods of time, and constant changes in the topography of land and altitude [4], with considerable positive and negative slopes [5], is also emphasized. Despite trail running races being extremely hard, the organization of the sport has exponentially grown worldwide in recent times [6–8], and Spain is no exception [9]. The marked demand expected today of such races has drawn more participants, to the extent that the number of runners in races must be limited, which has led to some runners becoming professionals with enhanced ultratrail race performance [10]. Int. J. Environ. Res. Public Health2021,18, 5119.

Int. J. Environ. Res. Public Health2021,18, 5119 2 of 14 Ultratrail races are multifactorial events that include physiological, neuromuscular, biomechanical and psychological variables [2]. Depending on exertion while running these races, research works indicate that according to ventilator thresholds (VT), in physiological terms these races place mean training intensities at 85.7% for zone I (<VT1), 13.9% for zone II (VT1–VT2) and 0.4% for zone III (>VT2) [11]. Cardiac responses during races lasting between 14 and 7 h present intensities of 64% [12] and 82% [13] of maximum heart rate (HRmax), respectively. Different research works have analyzed lactate kinetics during such races, and have obtained results that are below the onset of blood lactate accumulation (OBLA) [14,15]. Finally, as regards the psychophysiological component, values between 13 and 14 points have been obtained on the rate perceived exertion (RPE) scale [16]. In ultratrail endurance races, optimum performance might depend on high maximal oxygen consumption (VO2max) levels, high VO2maxfraction utilization percentages and low oxygen transport cost [2]. Along these lines, it has been demonstrated that maximum aerobic power can be a limiting factor to running on positive slopes [17], which are usual for ultratrail routes. When considering the types of routes that ultratrail races are organized along, it would appear that adapting to exertions, in which eccentric-type activations predominate, could be important for facing the ascents and constant loads that come into play when running on irregular sloping surfaces. The eccentric capacity to maintain maximum racing speed would be a determining factor in zones with negative slopes [18]. The increasing numbers of both participants and ultratrail races have favored different research studies being performed in an attempt to analyze the impact that this racing has on health. Indeed, ultratrail races have been associated with a negative energy bal- ance [13], muscle harm and in ammation [19,20], neuromuscular fatigue [21,22], cardiac dysfunctions, myocardial damage [13] and dehydration [1]. The relationship between hydration status and performance has been described [23,24]. Hydration status is directly related to physical performance [25]. Previous studies have reported the consequences of dehydration at the physical level, which can negatively affect

with a negative energy bal- ance [13], muscle harm and in ammation [19,20], neuromuscular fatigue [21,22], cardiac dysfunctions, myocardial damage [13] and dehydration [1]. The relationship between hydration status and performance has been described [23,24]. Hydration status is directly related to physical performance [25]. Previous studies have reported the consequences of dehydration at the physical level, which can negatively affect sports performance [26,27]. A small decrement in hydration status impaired physiologic function and performance during a trail running race [28]. In addition, dehydration status could negatively affect the neuromuscular response [29]. Bearing in mind the marked physical stress that the organism is submitted to while running an ultratrail race, it can be stated that running such races in an unplanned and uncontrolled manner can have very negative consequences for the health of the people practicing this sport [1]. Indeed, knowing the limits of the organism's physiological responses while practicing long intense exercise is necessary to avoid resorting to urgent medical assistance [30]. However, the impact of an ultratrail race could differ from one individual to another because they have distinct training levels. This means that more studies need to be conducted to elucidate this statement [31,32]. The impacts of such races could be very different depending on the participants' training levels, owing to their distinct physiological pro les [33]. Hence, the objectives of the present study were to: (a) evaluate the acute responses to an ultratrail race in terms of neuromuscular function, muscle damage and hydration status; (b) analyze if responses could differ according to training levels. 2. Materials and Methods 2.1. Participants Twenty runners participated in this quasiexperimental study, and were divided into an amateur-level group (n= 10) and a high-level group (n= 10) according to their training levels [34]. The high-level group was made up of runners who trained for a minimum of 8 h/week and achieved positive accumulated slopes of more than 50,000 m in the 12 months before the test. The amateur group was formed by those runners who did not meet any of the set requirements. The study was conducted on a population of 176

[34]. The high-level group was made up of runners who trained for a minimum of 8 h/week and achieved positive accumulated slopes of more than 50,000 m in the 12 months before the test. The amateur group was formed by those runners who did not meet any of the set requirements. The study was conducted on a population of 176 male participants who completed the race. The minimum sample size was 20 participants

Int. J. Environ. Res. Public Health2021,18, 5119 3 of 14 (con dence level = 95%; margin of error = 21%). The characteristics of the participants in each group are found in Table. Table 1.Characteristics of our participants. Parameters Amateur Level ( n= 10) High Level (n= 10) p Age (years) 43.30 4.52 41.40 6.18 0.443 Height (m) 1.77 0.06 1.76 0.06 0.780 Experience (year) 5.80 2.52 4.60 1.26 0.196 Weekly training (h) 6.50 0.70 11.05 2.94 <0.001 Annual slopes (m) 33,716.7 4427.7 56,426.6 8184.6 0.001 All the participants were informed about the study purpose and signed a consent form before enrolling. The protocol was reviewed and approved by the Ethical Committee of Clinical Research of Aragon (Spain) following the guidelines of the Helsinki Declaration of Ethics, updated at the World Medical Assembly in Fortaleza (2013) for research involving human subjects. A code was assigned to each participant to collect and treat samples in order to maintain their anonymity. In order to participate in this study, the participants had to meet the following inclusion criteria: (a) male; (b) nished the race route; (c) had no injuries after a race in at least the 2 months prior to the test; (d) not on a special diet or any pharmacological treatment; (e) no cardiovascular or metabolic diseases. 2.2. Procedures This study was carried out while organizing the Guara Somontano UltraTrail Race (GSUR) (Figure), which took place in the town of Alqu ²zar (Huesca, Spain). The total race distance was 108 km, with 5800 meters of positive accumulated slope. The maximum permitted time to nish it in was 24 h. During this race the mean temperature was 14 4.4 °C and relative humidity was 57 16.1% (Figure).Int. J. Environ. Res. Public Health 2021, 18, x 4 of 15 Figure 1. Race profile (taken from the organization’s website). 2.3. Anthropometric Measurements The participants’ morphological characteristics were evaluated in the morning and always under the same conditions. Body height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (Seca 220, Seca, Hamburg, Germany). Body weight was measured to the nearest 0.01 kg on calibrated

x 4 of 15 Figure 1. Race profile (taken from the organization’s website). 2.3. Anthropometric Measurements The participants’ morphological characteristics were evaluated in the morning and always under the same conditions. Body height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (Seca 220, Seca, Hamburg, Germany). Body weight was measured to the nearest 0.01 kg on calibrated electronic digital scales (Seca 769, Seca, Hamburg, Germany), barefooted. A Holtain© 610ND (Holtain, Crymych, Wales, UK) skin fold compass, accurate to ±0.2 mm and a tape (Seca 212, Seca, Hamburg, Germany) with an accuracy of ±1 mm were employed to take anthropometric assessments. The obtained anthropometric measurements were height, weight, six skin folds (abdominal, suprailiac, subscapular, tricipital, thigh and leg) and perimeters (arm and leg in a relaxed 90° posi- tion). The equations of Yushaz were used to calculate the percentage of fat [35] and the equation according to Lee to determine the percentage of muscle [36]. All the measure- ments were taken by the same operator, who was skilled in kinanthropometric techniques, and in accordance with the International Society for the Advancement of Kinanthropom- etry recommendations [37]. Body weight was recorded 2 h before and immediately after the race finished. 2.4. Physical Performance Evaluation In order to determine the corresponding physical performance values, a progressive and maximum laboratory test was done on a treadmill (Pulsar, h/p/cosmos®, Nussdorf, Germany). The test was run on a 1% slope and began at a speed of 8 km/h, which increased 1 km/h every minute. Before the test began, the participants warmed up for 5 minutes on the treadmill operating at a speed of 6 km.h -1 . Respired gases were collected with an Ox- ycon Pro analyzer (Erich Jaeger GmbH, Hoechberg, Germany). A pulsometer (Vantage M, Polar, Finland) was used to evaluate the maximal heart rate. 2.5. Neuromuscular Function Neuromuscular function was evaluated by different jumping tests: squat jump (SJ), countermovement jump (CMJ) and Abalakov jump (ABK) [38,39]. The former tests were chosen to measure the neuromuscular function of leg extensor muscles, because they can perform jumps with a high degree of reliability

pulsometer (Vantage M, Polar, Finland) was used to evaluate the maximal heart rate. 2.5. Neuromuscular Function Neuromuscular function was evaluated by different jumping tests: squat jump (SJ), countermovement jump (CMJ) and Abalakov jump (ABK) [38,39]. The former tests were chosen to measure the neuromuscular function of leg extensor muscles, because they can perform jumps with a high degree of reliability [40,41]. A jump mat system (Chronojump Boscosystems, Barcelona, Spain) was employed to measure the height and time during jumps. Three attempts were made for all jump types; there was a 30-second rest between jumps. The best jump was selected to be later analyzed. Evaluations were done 2 h before Figure 1.Race pro le (taken from the organization's website). The week before competition, the participants underwent body composition and physical performance evaluations. In parallel to these evaluations, the runners completed an openad hocsurvey with supplementary information about their experience, and the characteristics of their training over the past year (h/week and accumulated slope). To determine hydration during races, the volumes of liquids drunk by racers were calculated at both supply points and throughout the race according to individual drinking frequency.

Int. J. Environ. Res. Public Health2021,18, 5119 4 of 14 Volunteers recorded the amounts of water ingested at each supply point using two 250 cc bottles as a reference. At each refreshment point, two 250 cc bottles were given to each participant. 2.3. Anthropometric Measurements The participants' morphological characteristics were evaluated in the morning and always under the same conditions. Body height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (Seca 220, Seca, Hamburg, Germany). Body weight was measured to the nearest 0.01 kg on calibrated electronic digital scales (Seca 769, Seca, Hamburg, Germany), barefooted. A Holtain©610ND (Holtain, Crymych, Wales, UK) skin fold compass, accurate to 0.2 mm and a tape (Seca 212, Seca, Hamburg, Germany) with an accuracy of 1 mm were employed to take anthropometric assessments. The obtained anthropometric measurements were height, weight, six skin folds (abdominal, suprailiac, subscapular, tricipital, thigh and leg) and perimeters (arm and leg in a relaxed 90 position). The equations of Yushaz were used to calculate the percentage of fat [35] and the equation according to Lee to determine the percentage of muscle [36]. All the measurements were taken by the same operator, who was skilled in kinanthropometric techniques, and in accordance with the International Society for the Advancement of Kinanthropometry recommendations [37]. Body weight was recorded 2 h before and immediately after the race nished. 2.4. Physical Performance Evaluation In order to determine the corresponding physical performance values, a progressive and maximum laboratory test was done on a treadmill (Pulsar, h/p/cosmos®, Nussdorf, Germany). The test was run on a 1% slope and began at a speed of 8 km/h, which increased 1 km/h every minute. Before the test began, the participants warmed up for 5 min on the treadmill operating at a speed of 6 km.h 1 . Respired gases were collected with an Oxycon Pro analyzer (Erich Jaeger GmbH, Hoechberg, Germany). A pulsometer (Vantage M, Polar, Finland) was used to evaluate the maximal heart rate. 2.5. Neuromuscular Function Neuromuscular function was evaluated by different jumping tests: squat jump (SJ), countermovement jump (CMJ) and Abalakov jump (ABK) [38,39]. The

operating at a speed of 6 km.h 1 . Respired gases were collected with an Oxycon Pro analyzer (Erich Jaeger GmbH, Hoechberg, Germany). A pulsometer (Vantage M, Polar, Finland) was used to evaluate the maximal heart rate. 2.5. Neuromuscular Function Neuromuscular function was evaluated by different jumping tests: squat jump (SJ), countermovement jump (CMJ) and Abalakov jump (ABK) [38,39]. The former tests were chosen to measure the neuromuscular function of leg extensor muscles, because they can perform jumps with a high degree of reliability [40,41]. A jump mat system (Chronojump Boscosystems, Barcelona, Spain) was employed to measure the height and time during jumps. Three attempts were made for all jump types; there was a 30-s rest between jumps. The best jump was selected to be later analyzed. Evaluations were done 2 h before the race began and then immediately after the race nished (approximately 1 to 5 min after the race). The protocols of each jump test were applied by following the original protocol proposed by Bosco et al. [39]. In order to perform SJ, the participants started in a squatting position with knees bent at 90 and arms on hips to avoid in uencing the jump. A goniometer was used to verify the knee angle. The participants had to remain in this squatting position for 3 s before performing SJ. For CMJ, the subjects started from an upright standing position with hands on hips to avoid any arm movement. Then as a single sequence, they made a swift downward movement, followed immediately by a rapid vertical movement to jump as high as possible. Finally, during the ABK test, the participants had to begin by squatting and exing their knees 90 , followed by swinging their arms to help them to jump as high as possible. 2.6. Blood Samples Twenty milliliters of venous blood (antecubital vein) was withdrawn from each partic- ipant in both the pre- and post-race evaluations (90 min before and 10 min after nishing

to jump as high as possible. 2.6. Blood Samples Twenty milliliters of venous blood (antecubital vein) was withdrawn from each partic- ipant in both the pre- and post-race evaluations (90 min before and 10 min after nishing

Int. J. Environ. Res. Public Health2021,18, 5119 5 of 14 the race). Blood samples were collected in two 5-mL Vacutainer tubes (Vacutainer, beliver industrial state, plymouth PL6 7BP, United Kingdom) without anticoagulant for serum isolation and in two 5-mL tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Once collected, blood samples were coagulated for 25–30 min at room tem- perature and then centrifuged at 2500 rpm for 10 min to remove the clots. Serum samples were aliquoted into Eppendorf tubes (Eppendorf AG, Hamburg, Germany), previously washed with diluted nitric acid, and conserved at 80 °C until the biochemical analysis. 2.7. Determining Muscle Damage Markers and Hydration Status A 2-mL blood sample was used to determine leukocytes (leu) with an analyzer model Coulter model AcT diff. The rest, a 3-mL blood sample, was employed to determine creatinine (Cr), alanine aminotransferase (ALT), creatine kinase (CK), sodium (Na) and bilirubin (BIL) by spectrophotometric techniques. Complete biochemistry was processed in the laboratory of the San Jorge University Hospital by a Chemistry Analyzer model Advia 1650 (Bayer, Germany). 2.8. Statistical Analysis Data were processed with IBM SPSS 25.0 Statistics for Macintosh (IBM Corp., Armonk, NY, USA). A descriptive analysis was performed to show the means and standard devia- tions. The variables' normality distribution was analyzed by the Shapiro–Wilk test and the homogeneity of variances by the Levene test. The Student's t-test was applied to determine differences in the participants' characteristics and the percentages of change (pre-race vs. post-race). A two-way ANOVA (group effect + race effect) was used to show differences between the studied variables. Effect size was calculated for the two-way ANOVA using partial eta-squared ( 2) as a low effect (0.01–0.06), moderate effect (0.06–0.14) and high effect (>0.14) [42]. The p< 0.05 differences were considered statistically signi cant. 3. Results Table ferences existed in weekly training hours, annual slope achieved while training, times recorded during the GSUR test, maximum speed and percentage of fat (p< 0.05). Table 2.Body composition, physical tests and result during GSUR. Parameters Amateur Level (n= 10) High Level (n= 10) p Time during the race (h) 19.87 1.84 15.31

This study analyzes the effects of ultratrail running on muscle function and hydration in different training levels.