Effects of Beetroot Juice on Recovery of Muscle Function and Performance between Bouts of Repeated Sprint Exercise

This study examined the effects of beetroot juice (BTJ) on recovery between two repeated-sprint tests. In an independent groups design, 20 male, team-sports players were randomized to receive either BTJ or a placebo (PLA) (2 250 mL) for 3 days after an initial repeated sprint test (20 30 m; RST1) and after a second repeated sprint test (RST2), performed 72 h later. Maximal isometric voluntary contractions (MIVC), countermovement jumps (CMJ), reactive strength index (RI), pressure-pain threshold (PPT), creatine kinase (CK), C-reactive protein (hs-CRP), protein carbonyls (PC), lipid hydroperoxides (LOOH) and the ascorbyl free radical (A ) were measured before, after, and at set times between RST1 and RST2. CMJ and RI recovered quicker in BTJ compared to PLA after RST1: at 72 h post, CMJ and RI were 7.6% and 13.8% higher in BTJ vs. PLA, respectively (p< 0.05). PPT was 10.4% higher in BTJ compared to PLA 24 h post RST2 (p= 0.012) but similar at other time points. No group differences were detected for mean and fastest sprint time or fatigue index. MIVC, or the biochemical markers measured (p> 0.05). BTJ reduced the decrement in CMJ and RI following and RST but had no effect on sprint performance

was 10.4% higher in BTJ compared to PLA 24 h post RST2 (p= 0.012) but similar at other time points. No group differences were detected for mean and fastest sprint time or fatigue index. MIVC, or the biochemical markers measured (p> 0.05). BTJ reduced the decrement in CMJ and RI following and RST but had no effect on sprint performance or oxidative stress. Keywords:beetroot juice; muscle damage; exercise recovery; repeated sprint exercise 1. Introduction Repeated sprint exercise (RSE), in which a number of short-duration maximal effort sprints (2–6 s) are completed intermittently with brief recovery periods ( 60 s), places a great deal of stress on the physiological and musculoskeletal systems [1]. The high energy turnover during RSE induces signi cant metabolic stress, triggering rapid perturbations in the nervous, immune, and endocrine systems [1], as well as an increased formation of reactive oxygen species (ROS) [2]. In addition, the high-force eccentric muscle contractions required to accelerate and decelerate during RSE places a great deal of mechanical stress on the musculoskeletal system, particularly the quadriceps and hamstring muscle groups [3]. It is therefore not surprising that team-sport players, who routinely engage in RSE in training sessions and matches, often display symptoms of muscle damage (i.e., muscle soreness and reduced muscle function) that can persist for several days [4–6]. Because the typical time between Nutrients2016,8, 506; doi:10.3390/nu8080506

Nutrients2016,8, 506 2 of 17 training sessions and or matches is often not suf cient for full recovery ( 72 h) athletes and coaches are continually seeking strategies that could help minimise the negative effects of muscle damage [7,8]. The exact mechanisms to explain the causes of muscle damage after RSE are not fully understood, but a host of factors such as muscle membrane damage, sarcomere disorganization, excitation-contraction coupling dysfunction, contractile protein degradation and in ammation are all likely to play a role [9,10]. Furthermore, it has been suggested that the generation of ROS in the days post-exercise, likely a consequence of in ammatory mediated repair processes, might exacerbate the existing muscle damage by degrading components of the cytosol that are integral to force production [11–13]. A number of studies have provided evidence of oxidative stress in the hours and days following RSE [2,6,14], suggesting that the endogenous antioxidant system is unable to cope with excess ROS production under these conditions. Thus, it would be reasonable to assume that the prolonged decrement in muscle function might be, at least in part, attributable to oxidative stress. This also makes the expectation tenable that interventions attempting to combat the excess production of ROS and control oxidative stress, such as antioxidants, could help accelerate the rate of muscle recovery following RSE. While the nutritional antioxidants vitamin C and E have proven largely ineffective at attenuating muscle damage [15–17], there is growing support for the use of antioxidant-rich fruit and vegetable beverages as recovery aids [18]. Recently, we showed that supplementation with beetroot juice (BTJ) attenuated some aspects of muscle damage following high intensity plyometric exercise [19]. We proposed that one of the potential mechanisms by which of BTJ might have attenuated EIMD in this study was via its antioxidant effects. Although the antioxidant effects of BTJ has received little attention in the literature, ndings from our previous work [20] and others [21,22] suggest that its antioxidant capacity is markedly higher than other vegetable juices, such as tomato and carrot juice, and also than several other drinks considered to have a high antioxidant capacity

this study was via its antioxidant effects. Although the antioxidant effects of BTJ has received little attention in the literature, ndings from our previous work [20] and others [21,22] suggest that its antioxidant capacity is markedly higher than other vegetable juices, such as tomato and carrot juice, and also than several other drinks considered to have a high antioxidant capacity such as green tea, apple, cherry and cranberry juice [20,23,24]. The high antioxidant capacity of BTJ is due to the fact that several of the phytonutrient compounds it contains have been shown to scavenge ROS productionin vitro andin vivoand subsequently limit cellular injury [25–27]. The most potent antioxidant molecules in BTJ are thought to be the betalain pigments, which are responsible for beetroot's violet colour [28]. The betalains, and betanin in particular, are very effective electron donors that have been shown to not only attenuate ROS mediated injury but also to upregulate endogenous antioxidant enzymes and stimulate host defence [29–32]. In addition, BTJ is rich in nitrate, which, via its reduction to nitric oxide (NO) might have indirect antioxidant effects by supressing the accumulation of leukocytes [33], which are thought to be the main producers of ROS after muscle-damaging exercise [34]. Nonetheless, we failed to quantify oxidative stress in our aforementioned experiment [19] to con rm or refute this posit. Furthermore, the aforementioned ndings were in recreationally active participants and therefore these results might not be directly transferable to better trained athletic populations. To our knowledge, the effects of BTJ on muscle damage and recovery after activity incorporating RSE has not been investigated. Additionally, the effectiveness of such an intervention on subsequent performance has not been considered. Therefore, the main aim of this study was to examine whether BTJ can attenuate losses in muscle function and performance between two sport-speci c repeated sprint tests (RST) performed 72 h apart. We also examined the effects of BTJ on biochemical markers associated with muscle damage, speci cally oxidative stress, to try and discern what role (if any) the antioxidant potential of BTJ has on attenuating EIMD. Based on our previous ndings [19],

attenuate losses in muscle function and performance between two sport-speci c repeated sprint tests (RST) performed 72 h apart. We also examined the effects of BTJ on biochemical markers associated with muscle damage, speci cally oxidative stress, to try and discern what role (if any) the antioxidant potential of BTJ has on attenuating EIMD. Based on our previous ndings [19], we hypothesized that: (1) BTJ would attenuate muscle function de cits and oxidative stress between and after the two repeated sprint tests; and (2) that performance during the second sprint test would be preserved with BTJ compared to a placebo.

Nutrients2016,8, 506 3 of 17 2. Materials and Methods 2.1. Participants Twenty male participants gave written informed consent for participation in this study (characteristics presented in Table). The sample size for this study was based on a priori power calculation. Based on a previous study [19], with a power of 0.80 and two tailed level set at 0.05, the minimum number of participants required to detect an 8% difference in counter movement jump (CMJ) performance between groups (SD: 6%) was estimated as 10 per group. We selected CMJ as our primary outcome measure because it is believed to be the most sensitive test for detecting reductions in neuromuscular function after RSE [35]. Our secondary outcomes included other markers of neuromuscular function, repeated sprint performance, muscle pain, and biochemical markers of in ammation, oxidative stress and muscle damage (speci c details in relevant sections below). All participants were collegiate team-sports players, competing in either soccer (n= 10), rugby (n= 5), basketball (n= 2) hockey (n= 2) or handball (n= 1) on a regular basis; all testing was performed at the end of the competitive season (between March 2015 and June 2015). Participant's eligibility was assessed with a health screening questionnaire. None had any known food allergies, were suffering from a musculoskeletal injury, or had previous history of renal, gastrointestinal or cardiovascular complications or any other contraindication to the study procedures. For the 48 h prior to and throughout data collection, participants were prohibited from consuming alcoholic beverages, and instructed to avoid any strenuous exercise outside of the trial requirements. The study protocol received ethical approval from the Faculty of Health and Life Sciences ethics committee at Northumbria University. Approval was granted on the 26 February 2015 and assigned the following project identi cation code: HLSTC200115. Table 1.Descriptive data for participants in the beetroot juice (BTJ) and placebo (PLA) groups. Group Age (Years) Height (m) Mass (kg) BTJ 23 3 1.83 0.90 76.8 9.5 PLA 21 2 1.77 0.51 73.4 12.4 Values are mean SD (n= 10 per group). No signi cant differences were detected between groups for any variable (p> 0.05).

identi cation code: HLSTC200115. Table 1.Descriptive data for participants in the beetroot juice (BTJ) and placebo (PLA) groups. Group Age (Years) Height (m) Mass (kg) BTJ 23 3 1.83 0.90 76.8 9.5 PLA 21 2 1.77 0.51 73.4 12.4 Values are mean SD (n= 10 per group). No signi cant differences were detected between groups for any variable (p> 0.05). 2.2. Experimental Design This study employed a double-blind, placebo controlled, independent groups design. Participants were required to attend the laboratory for 6 visits over a 2 week period. The rst visit was to familiarise the participants with the study procedures and randomly allocate them to either a beetroot juice (BTJ) or an isocaloric placebo (PLA) group. Their baseline maximal isometric voluntary contraction (MIVC) was used to match the groups. The principal investigator was responsible for the randomizing procedures. The next ve visits were performed on consecutive days in the same laboratory at the same time of day and were preceded by an overnight fast. For the main trials, participants performed two repeated sprint tests separated by 72 h (visit 2 = RST1 and visit 5 = RST2) (see Figure schematic outline). A range of dependent variables were taken pre, 30-min post, 24, 48 and 72 h after RST1, and 30-min post and 24 h after RST2 to monitor recovery. On each occasion, dependent variables were performed in the following order: pressure-pain threshold (PPT), venous blood draw, CMJ, reactive strength index (RI) and MIVC. After completing the post-exercise measures participants consumed 1 serving of their allocated treatment, and returned to the lab 2.5 h post ingestion for a further blood sample. Another treatment was taken with an evening meal, and then at the same points (with breakfast and with an evening meal) for the following 3 days. All data collection took place in the exercise laboratories at Northumbria University.

Nutrients2016,8, 506 4 of 17Nutrients 2016, 8, 506 4 of 16 Figure 1. Schematic outline of study procedures. 2.3. Repeated Sprint Test The RST consisted of 20 maximal‐effort 30 m sprints, interspersed by 30 s of passive recovery. A 10 m deceleration zone was marked out at the end of each 30 m sprint, in which participants were required to stop within; the 30 s rest period commenced when participants had come to a halt. The RST was adapted from previous studies that showed repeated sprints with forced decelerations induce substantial muscle damage and fatigue in team‐sport trained participants [3,36,37]. Furthermore, the muscle damage induced by an analogous RST seems to cause reductions in muscle function not different to those observed after intermittent sport simulations [38] and competitive matches [39]. Before performing each RST, participants undertook a standardized warm up as previously described [3]. Briefly, participants completed 400 m of self‐selected jogging, a series of dynamic stretches, and sprints at 60% and 80% of maximal effort. Participants were then given a further 5 min to complete their own stretching. Timing gates (Brower Timing Systems, Draper, UT, USA) were positioned at 0 and 30 m to record sprint times. Participants were instructed to give maximal‐effort for each sprint and were provided with strong verbal encouragement throughout. All testing took place in an air conditioned sprint track in similar environmental conditions. 2.4. Maximal Isometric Voluntary Contractions MIVC of the right knee extensors was assessed as previously described [3,19]. Participants were seated and fitted to a portable strain gauge (MIE Medical Research Ltd., Leeds, UK) via a plinth placed just above the malleoli of the right ankle. In this positon, joint angle was adjusted to 90° of knee flexion using a goniometer and marked to ensure consistency across visits. Participants performed 3 maximal effort isometric contractions, each lasting 3 s, and separated by 60 s seated rest. The peak value in Newton’s (N) was used for analysis. Coefficient of variation (CV) for this protocol in our lab was calculated as 1.1%. 2.5. Counter Movement Jump CMJ height was determined from flight time using

marked to ensure consistency across visits. Participants performed 3 maximal effort isometric contractions, each lasting 3 s, and separated by 60 s seated rest. The peak value in Newton’s (N) was used for analysis. Coefficient of variation (CV) for this protocol in our lab was calculated as 1.1%. 2.5. Counter Movement Jump CMJ height was determined from flight time using an optical measurement system (Optojump next, Bolzano, Italy). Participants started the movement upright with hands fixed to their hips and after a verbal cue, descended into a squat prior to performing a maximal effort vertical jump. Participants performed 3 maximal efforts, separated by 30 s standing recovery. Mean height (cm) was used for analysis. The CV for this protocol in our lab was calculated as 2.1%. Figure 1.Schematic outline of study procedures. 2.3. Repeated Sprint Test The RST consisted of 20 maximal-effort 30 m sprints, interspersed by 30 s of passive recovery. A 10 m deceleration zone was marked out at the end of each 30 m sprint, in which participants were required to stop within; the 30 s rest period commenced when participants had come to a halt. The RST was adapted from previous studies that showed repeated sprints with forced decelerations induce substantial muscle damage and fatigue in team-sport trained participants [3,36,37]. Furthermore, the muscle damage induced by an analogous RST seems to cause reductions in muscle function not different to those observed after intermittent sport simulations [38] and competitive matches [39]. Before performing each RST, participants undertook a standardized warm up as previously described [3]. Brie y, participants completed 400 m of self-selected jogging, a series of dynamic stretches, and sprints at 60% and 80% of maximal effort. Participants were then given a further 5 min to complete their own stretching. Timing gates (Brower Timing Systems, Draper, UT, USA) were positioned at 0 and 30 m to record sprint times. Participants were instructed to give maximal-effort for each sprint and were provided with strong verbal encouragement throughout. All testing took place in an air conditioned sprint track in similar environmental conditions. 2.4. Maximal Isometric Voluntary Contractions MIVC

complete their own stretching. Timing gates (Brower Timing Systems, Draper, UT, USA) were positioned at 0 and 30 m to record sprint times. Participants were instructed to give maximal-effort for each sprint and were provided with strong verbal encouragement throughout. All testing took place in an air conditioned sprint track in similar environmental conditions. 2.4. Maximal Isometric Voluntary Contractions MIVC of the right knee extensors was assessed as previously described [3,19]. Participants were seated and tted to a portable strain gauge (MIE Medical Research Ltd., Leeds, UK) via a plinth placed just above the malleoli of the right ankle. In this positon, joint angle was adjusted to 90 of knee exion using a goniometer and marked to ensure consistency across visits. Participants performed 3 maximal effort isometric contractions, each lasting 3 s, and separated by 60 s seated rest. The peak value in Newton's (N) was used for analysis. Coef cient of variation (CV) for this protocol in our lab was calculated as 1.1%. 2.5. Counter Movement Jump CMJ height was determined from ight time using an optical measurement system (Optojump next, Bolzano, Italy). Participants started the movement upright with hands xed to their hips and after a verbal cue, descended into a squat prior to performing a maximal effort vertical jump. Participants performed 3 maximal efforts, separated by 30 s standing recovery. Mean height (cm) was used for analysis. The CV for this protocol in our lab was calculated as 2.1%.

Nutrients2016,8, 506 5 of 17 2.6. Reactive Strength Index Reactive strength index (RI) was used to measure the impact of muscle damaging exercise on participant's ability to utilize the stretch shortening cycle and perform explosive actions. In a similar fashion to previous studies [40], participants performed a drop jump from a 30 cm box and, upon landing, immediately jumped vertically, with instructions to minimise ground contact time while maximising jump height. RI was calculated as jump height divided by ground contact time (cm/ms) recorded from an optical measurement system (Optojump next). Participants performed 3 maximal efforts separated by 30 s of passive (standing recovery) with the mean height of the 3 jumps used for analysis. The CV for this protocol was calculated as 1.9% in our lab. 2.7. Treatments and Dietary Control Participants consumed 2 bottles (250 mL per bottle) of their assigned treatment (BTJ or PLA) on the day, 24, and 48 h after RST1 and 30-min post RST2, equating to 8 servings in total. One bottle was consumed 30-min after each trial, and one with an evening meal. The BTJ was supplied by Gs Fresh Ltd., (Cambridgeshire, UK) and consisted of 99% beetroot juice concentrate, nitrate and other phytonutrients; speci c details of the antioxidant capacity and phytonutrient content of this drink can be found elsewhere [20]. The PLA consisted of a low fruit containing (<1%) squash (Kia Ora, Coca Cola Enterprises, Uxbridge, UK), avourless protein powder (Arla Foods, Amba, Denmark) and maltodextrin powder (Myprotein, Manchester, UK) providing a negligible amount of phytochemicals and nitrate. Treatments were closely matched for volume, macro-nutrient and energy content, but differed in antioxidant capacity and nitrate content (see Table). Participants were provided with food dairies to record their intake 24 h prior to RST1 up until data collection was complete (24 h post RST2; 5 days in total). Average energy and macronutrient intake for each group is presented in Table. To comply with the double-blind, randomized design, drinks were provided in identically masked bottles, only distinguished by a single letter code. These were prepared by an individual not involved in data collection.

to RST1 up until data collection was complete (24 h post RST2; 5 days in total). Average energy and macronutrient intake for each group is presented in Table. To comply with the double-blind, randomized design, drinks were provided in identically masked bottles, only distinguished by a single letter code. These were prepared by an individual not involved in data collection. As detailed in a previous study [19], due to the distinct taste of BTJ, the PLA was not matched for taste and texture, only energy content. While others have used nitrate depleted BTJ as a PLA so that the taste is the same, this is not a true PLA because it will still contain many other bioactive constituents (i.e., phenolics and betalains) that, as outlined in the introduction, could favourably affect recovery. Thus, this would not have been plausible in the present study. Rather, in an attempt to overcome this, the participants were not informed of what the speci c drinks being investigated were. The only information they received was that they were antioxidant-containing drinks used for recovery. This ensured that the participants did not know the overall aim of the study, eliminating any bias based on pre-conceptions regarding BTJs potential ergogenic effects. Additionally, because we employed an independent groups design, participants were never aware of the taste/texture of the other treatment under investigation. Table 2. Energy and macronutrient content, trolox equivalence antioxidant capacity (TEAC) and nitrate content of the beetroot juice (BTJ) and placebo (PLA) supplements. Treatment BTJ PLA Energy (Kcals) 81 77 Volume (mL) 250 250 Carbohydrate (g) 16.4 16.4 Protein (g) 2.8 2.8 Fat (g) 0.4 Trace Nitrate (mg) 143 Trace TEAC * (mmol L 1 ) 11.4 0.2 0.25 0.02 * Estimation based on previous analyses [20].

The study investigates beetroot juice's impact on muscle recovery in team-sport athletes.