BETA ALANINA the Effect of Β-Alanine Supplementation During High-Intensity Interval Training on 3 Repeated Sprint Ability Performance and Neuromuscular Fatigue

1 TITLE
2 The effect of β-alanine supplementation during high-intensity interval training on
3 repeated sprint ability performance and neuromuscular fatigue
5 RUNNING HEAD: β-Alanine during HIIT improves performance
7 AUTHORS
8 Fabio Milioni1, Rodrigo Araújo Bonetti de Poli1, Bryan Saunders2,3, Bruno Gualano2, Alisson
9 Luiz da Rocha4, Adelino Sanchez Ramos da Silva4, Paulo de Tarso Guerrero Muller5,
10 Alessandro Moura Zagatto1,6*.
11
12 AFFILIATION & ADDRESSES
1
13 Post Graduate Program in Human Movement Sciences, São Paulo State University
14 (UNESP), Laboratory of Physiology and Human Performance, Bauru, SP, Brazil
2
15 Applied Physiology and Nutrition Research Group, School of Physical Education and Sport;
16 Rheumatology Division; Faculdade de Medicina FMUSP, University of São Paulo, Sao
17 Paulo, SP, Brazil
3
18 Institute of Orthopaedics and Traumatology, Faculty of Medicine FMUSP, University of São
19 Paulo, Brazil.
4
20 School of Physical Education and Sports of Ribeirão Preto (EEFERP), University of São
21 Paulo (USP), Ribeirão Preto, SP, Brazil
Downloaded from www.physiology.org/journal/jappl at Univ of Florida (128.227.024.141) on October 19, 2019.

5
22 Laboratory of Respiratory Pathophysiology (LAFIR), Mato Grosso do Sul Federal
23 University (UFMS), Campo Grande, MS, Brazil
6
24 São Paulo State University (UNESP), Faculty of Sciences, Department of Physical
25 Education, Bauru, SP, Brazil
26 *Corresponding author: São Paulo State University (UNESP), Faculty of Sciences,
27 Department of Physical Education, Laboratory of Physiology and Sport Performance.
28 Av. Luiz Edmundo Carrijo Coube,14-01, Vargem Limpa, CEP 17033-360 – Bauru/SP,
29 Brazil.
30 E-mail: azagatto@yahoo.com.br Phone: +55 (14) 31039628
31

32 ABSTRACT
33 The study investigated the influence of β-Alanine supplementation during a high-intensity
34 interval training (HIIT) program on repeated sprint ability (RSA) performance. This study
35 was randomized, double-blinded and placebo-controlled. Eighteen men performed an
36 incremental running test until exhaustion (TINC) at baseline and followed by 4-week HIIT
37 (10×1-min runs 90% maximal TINC velocity [1-min recovery]). Then, participants were
38 randomized into 2 groups and performed a 6-week HIIT associated with supplementation of
39 6.4g·day-1 of β-Alanine (Gβ) or dextrose (Placebo group; GP). Pre and Post 6-week
40 HIIT+supplementation, participants performed the following tests: 1) TINC; 2) supramaximal
41 running test; and 3) 2×6×35-m sprints (RSA). Before and immediately after RSA,
42 neuromuscular function was assessed by vertical jumps, maximal isometric voluntary
43 contractions of knee extension and neuromuscular electrical stimulations. Muscle biopsies
44 were performed to determine muscle carnosine content, muscle buffer capacity in vitro
45 (βminvitro) and content of phosphofructokinase (PFK), monocarboxylate transporter 4 (MCT4)
46 and hypoxia-inducible factor-1α (HIF-1α). Both groups showed a significant time-effect for
47 maximal oxygen uptake (Gβ:6.2±3.6% and GP:6.5±4.2%; p>0.01); only Gβ showed a time
48 effect for total (-3.0±2.0%; p=0.001) and best (-3.3±3.0%; p=0.03) RSA times. A group-by-
49 time interaction was shown after HIIT+Supplementation for muscle carnosine
50 (Gβ:34.4±2.3mmol.kg-1dm and GP:20.7±3.0mmol.kg-1dm; p=0.003) and neuromuscular
51 voluntary activation after RSA (Gβ:87.2±3.3% and GP:78.9±12.4%; p=0.02). No time effect
52 or group-by-time interaction were shown for supramaximal running test performance,
53 βminvitro and content of PFK, MCT4 and HIF-1α. In summary, β-Alanine supplementation
54 during HIIT increased muscle carnosine and attenuated neuromuscular fatigue, which may
55 contribute to an enhancement of RSA performance.
56 Keywords: Anaerobic capacity, muscle carnosine, muscle buffer capacity, western blot

57 New & Noteworthy: β-Alanine supplementation during a HIIT program increased repeated
58 sprint performance. The improvement of muscle carnosine content induced by beta-alanine
59 supplementation may have contributed to an attenuation of central fatigue during repeated
60 sprint. Overall, β-Alanine supplementation may be a useful dietary intervention to prevent
61 fatigue.

62 INTRODUCTION
63 Beta (β)-Alanine intake is common in recreational exercise practitioners to high level athletes
64 (46) and is an important pre-cursor in muscle carnosine (β-alanyl-L-histidine) synthesis (40).
65 Muscle carnosine is a dipeptide that performs several physiological roles including
66 antioxidant scavenging, regulation of calcium sensitivity, and muscle buffering due its acid
67 dissociation constant of 6.83 that makes it an efficient acceptor of hydrogen ions (for review,
68 see (2)). Supplementation of 4.0 – 6.4 g.day-1 of β-Alanine during 4 – 24 weeks can increase
69 muscle carnosine content (21,22,41) and muscle buffer capacity, although few studies have
70 directly measured the latter (21,22). Thus, supplementation of β-Alanine may be a promising
71 strategy to minimize muscle acidosis (23,40) induced by repeated all-out sprints.
72 The ability to repeatedly perform all-out sprints interspaced by incomplete recovery (termed
73 repeated sprint ability; RSA) is a fitness component of several sports modalities (6) and
74 results in hydrogen accumulation and a drop in muscle pH (6,32). This proton accumulation
75 may inhibit anaerobic glycolysis since in vitro analysis have verified that
76 phosphofructokinase (PFK - key regulatory glycolytic enzyme) activity is downregulated by
77 a pH decrement (47,48). In addition, muscle acidosis can impair the neuromuscular system
78 (17) and has been suggested as an important trigger of the group III/IV muscle afferent
79 feedback which is responsible for restraining the central motor drive and prevent a critical
80 threshold of peripheral fatigue to be reached (1,25).
81 This impairment of anaerobic glycolytic activity (28,47,48) and subsequent neuromuscular
82 fatigue induced (17) by muscle acidosis may impair RSA performance. Despite the potential
83 to increase muscle buffering capacity, the effect of β-Alanine supplementation on RSA
84 performance, as well as the likely mechanisms involved, are unexplored. Sweeney et al. (43),
85 Ducker et al. (13) and Milioni et al. (33), showed no ergogenic effect of β-Alanine

86 supplementation on RSA performed in running (non-motorized treadmill, running track and
87 sprints with change of direction), while Brisola et al. (8) and Claus et al. (10) observed
88 discrete improvement on RSA performed in swimming by professional and young water polo
89 players.
90 Theoretically, the potential to delay acidosis due to increased muscle buffering from muscle
91 carnosine would allow a greater effectiveness of anaerobic glycolysis, improving
92 performance and consequently generating a higher blood lactate concentration. Recent
93 evidence suggests that lactate is a powerful signaling molecule, able to alter the expression of
94 PFK (28,29) and upregulate hypoxia-inducible factor-1α (HIF-1α) (16,35), which can
95 increase anaerobic glycolysis (35) and lactate production and transport (16,35), through the
96 expression of the monocarboxylate transporter 4 (MCT4) (35,45). Similarly, improved
97 capacity to handle intramuscular hydrogen accumulation would protect against
98 neuromuscular fatigue. However, there is no information regarding which mechanisms at the
99 biomolecular level would be modulated by β-Alanine supplementation, especially when
100 administered concomitantly with a training program, and its potential contribution to
101 performance.
102 High-intensity interval training (HIIT) is an efficient tool to improve anaerobic capacity
103 (9,44) as well as RSA performance (27). Although the mechanisms for this improved
104 anaerobic capacity and RSA performance induced by HIIT are not completely clear, there
105 may be a link between HIIT and the increment of muscle carnosine content (12) and buffer
106 capacity (15). Also, studies investigating the practical relevance of β-Alanine
107 supplementation during controlled training routines (i.e., HIIT) and the possible benefits of
108 the association of both strategies (HIIT+supplementation) reported likely beneficial effects
109 (5,19,42).

110 We hypothesized that β-Alanine supplementation during HIIT would increase the muscle
111 carnosine content and consequently muscle buffer capacity, allowing greater molecular
112 adaptation of PFK muscle content (i.e. key regulatory glycolytic enzyme) system during the
113 RSA. Also, the increased capacity to handle hydrogen accumulation during a high intensity
114 effort (i.e., RSA) would protect against neuromuscular fatigue contributing to improve RSA
115 performance. Thus, the present study investigated whether β-Alanine supplementation
116 administered during HIIT could: 1) increase the muscle carnosine content and muscle
117 buffering capacity; 2) alter the content of PFK, MCT4 and HIF-1α; 3) attenuate
118 neuromuscular fatigue; and 4) enhance RSA performance.
119
120 METHODS
121 Participants
122 The sample size was calculated using G*Power software (University of Düsseldorf,
123 Germany), based on the assumption that 4 weeks of 6.4 g.day-1 of β-Alanine supplementation
124 can increase muscle carnosine content with an effect size of 1.96 (41). Using a statistical
125 power of 95% and alpha level of 0.05, the sample size calculated was n = 9 participants in
126 each group. Thus, 18 physically active males, were recruited to be part of the investigation
127 (initial maximal oxygen uptake (Vሶ Omax ): mean ± standard deviation (SD) 43.7 ± 3.8 mL.kg-
1
128 .min-1, body weight: 74.3 ± 8.4 kg, height: 174.8 ± 6.4 cm; age: 25 ± 5 years). Exclusion
129 criteria included non-omnivore (i.e., vegetarian or vegan), regular user (in the last 6 months)
130 of creatine, β-Alanine and/or whey protein and presence of any musculoskeletal disorder. The
131 participants were asked to maintain their nutritional habits during participation in the study,
132 as well as to abstain from strenuous activities and caffeine for 24-h and consume a light meal
133 2-h before each training/testing session. The study was approved by the Local Ethics

134 Committee and was conducted according to the Declaration of Helsinki. All participants were
135 informed about the procedures, benefits and risks of the investigation before signing an
136 informed consent form prior to beginning the study (Figure 1).
137
138 ***** Figure 1 near here*****
139
140 Experimental design
141 The participants first performed an incremental running test until exhaustion (TINC) to
142 determine Vሶ Omax and maximal aerobic velocity. Thereafter, participants initiated a 4-week
143 HIIT program detailed below. The 4-week training stimulus, without supplementation, aimed
144 to induce an initial positive performance adaptation to avoid HIIT overwhelming the potential
145 effects of β-Alanine supplementation as suggested by Cochran et al. (11). After 4-week of
146 training, participants were matched for Vሶ Omax and randomized into a β-Alanine (Gβ: 6.4
147 g.day-1 of β-Alanine; n = 9) or Placebo (GP: 6.4 g.day-1 of dextrose; n = 9) group and
148 continued the HIIT-based training program associated with the supplementation protocol
149 (HIIT+supplementation) during a further 6 weeks. Pre and Post HIIT+supplementation, the
150 participants performed the following tests with 48-h recovery between them: 1) TINC; 2)
151 supramaximal running test until exhaustion (TSUPRA); and 3) 2 bouts of 6×35-m all-out sprints
152 (RSA). Before (i.e., resting condition) and immediately after (i.e., fatigued condition) RSA,
153 neuromuscular function was assessed by maximum vertical counter movement jumps,
154 maximal isometric voluntary contractions (MVC) of the knee extensors and peripheral
155 neuromuscular electrical stimulations. All protocols (except RSA) were performed on a
156 stationary treadmill (ATL, Inbramed, Porto Alegre, Brazil), with 5 min warm-up at 8 km.h-1
157 and 5-min of passive recovery prior to the effort. Participants underwent muscle biopsies at

158 Pre and Post HIIT+supplementation. 48 – 72 h following the final physical test to determine
159 muscle carnosine content, muscle buffering capacity in vitro (βmin vitro) and the content of
160 PFK, MCT4 and HIF-1α. (Figure 2).
161
163
164 Incremental running test (TINC)
165 The protocol started at 8 km.h-1 with increments of 1.5 km.h-1 every 2 min until volitional
166 exhaustion. Vሶ Omax was considered to be the highest average of the oxygen uptake obtained
167 during the final 30 s of each stage, assuming the oxygen uptake plateau (i.e., range < 2.1
168 mL.kg-1.min-1 in the final 2 stages) as primary criteria and secondarily a respiratory exchange
169 ratio > 1.10, maximal heart rate > 90% of maximum predicted HR (220 – age) and peak
170 blood lactate concentration ≥ 8.0 mmol.L-1 (24). Maximal aerobic velocity was determined as
171 the highest velocity achieved during TINC, however, when the participant remained less than
172 30 s in the final stage, maximal aerobic velocity was adjusted according to Kuipers et al. (26).
173
174 High Intensity interval training (HIIT)
175 The HIIT sessions consisted of 10 × 1-min runs at 90% of maximal aerobic velocity with 1-
176 min passive recovery (adapted from Little et al. (30)). Heart rate (Polar RS400, Kempele,
177 Finland) was measured during each run and participants were required to reach 90% of
178 maximal heart rate in the last 5 runs; if they did not, the intensity was increased by ~3% of
179 maximal aerobic velocity in the subsequent HIIT session. Eleven training sessions were
180 performed during the initial 4-week of HIIT and 17 during the 6-week

181 HIIT+supplementation, with 36 - 72 h rest between sessions. All training sessions were
182 supervised by a member of the research team and participants presented excellent adherence
183 to training program with 98% of all sessions completed.
184
185 Supplementation protocol
186 Participants ingested 6.4 g.day-1 of β-Alanine (CarnoSyn β-Alanine, NAI, USA) or placebo
187 (unflavored dextrose, Max Titanium, Supley, Matley, SP, Brazil) administered orally in 800
188 mg gastro-resistant capsules coated with hydroxypropyl-methylcellulose (DRcaps, Capsugel,
189 France). Participants were suggested to ingest supplements with meals (two capsules per
190 meal) to avoid paraesthesia (23,41).
191 In the fifth week of supplementation, one participant (belonging to GP) was excluded from
192 the study due to the impossibility of maintaining the supplementation protocol, and the
193 remaining participants self-reported 100% of adherence to the supplementation protocol.
194
195 Supramaximal running test (TSUPRA)
196 The supramaximal running test was performed at 115% of previously determined individual
197 maximal aerobic velocity and participants were required to exercise until exhaustion. Oxygen
198 uptake averaged over the final 30 s of the test and time-to-exhaustion were recorded as
199 performance indicators. Blood lactate concentration was also measured.
200
201 Repeated Sprint Ability Test (RSA)

202 The participants performed the Running-Based Anaerobic Sprint Test (RAST) (Intraclass
203 correlation of RAST variables ~0.88 (49)) twice, with 4-min of passive recovery between
204 sets. The RAST consists of 6 × 35-m all-out sprints with 10-s passive recovery between
205 sprints. All sprints were recorded by two video cameras (GoPro Hero 3+ Black, San Mateo,
206 CA, USA) synchronized and positioned laterally to the track. Optical sensors which emitted
207 light and a “beep” sound when the participants passed through were positioned at the finish
208 lines. Sprint times were analyzed using Kinovea software (Kinovea 0.8.15 for Windows) and
209 the variables measured from RSA were total time, best time, mean time, worst time and
210 fatigue index [100 × (total time / (best time × 12) – 100].
211
212 Physiological variables
213 Throughout the TINC and TSUPRA, oxygen uptake was measured breath-by-breath using an
214 ergospirometer (Quark PFT, Cosmed, Rome, Italy) calibrated prior to each test according to
215 standard procedures. For oxygen uptake analysis, data were smoothed every 5 points and
216 interpolated every 1 s to eliminate outlying data. Heart rate was measured using a transmitter
217 belt coupled to the gas analyzer (Wireless HR 138 monitor; Cosmed, Rome, Italy). Blood
218 samples (25 µL) were collected from the earlobe pre-exercise and 3, 5 and 7 min following
219 exercise (TINC, TSUPRA and RAST), stored in 1.5 mL plastic tubes containing 50 µL of sodium
220 fluoride (1%) and immediately frozen at -20°C for later analyses using an electrochemical
221 analyzer (YSI 2900, Yellow Spring Instruments, Yellow Spring, OH, USA) (measurement
222 error: ±2%).
223
224 Neuromuscular function assessments (NMA)
225 Vertical jumps

226 Three counter movement jumps, with 1-min rest between jumps, were performed on a jump
227 platform (Jump test, CEFISE, Nova Odessa, SP, Brazil), and the highest jump attempt of
228 each participant was used. The intraclass correlation and coefficient of variation for jump
229 height were 0.90 (0.80 – 0.95) and 5.3%.
230 MVC measurements and femoral nerve electrical stimulations
231 One minute after the final jump, participants performed three MVCs separated by 1-min rest.
232 A custom-design chair maintained a 90° flexion of participants’ hips and knees; participants
233 were firmly fixed to the chair with straps across the chest, hip and thigh. The ankle of the
234 dominant leg was attached to a load cell (SDK200, Miotec, Porto Alegre, RS, Brazil) and the
235 force signal of MVC was acquired at 2000 Hz. Supramaximal, square-wave, electrical pulses
236 were delivered on the femoral nerve by a constant current electrical stimulator
237 (Bioestimulador, Insigth, Ribeirão Preto, SP, Brazil) (400 V max). Electrodes (5 × 5 cm) with
238 conductive gel were placed in the femoral triangle (cathode) and the gluteal fold (anode), and
239 marked with ink for precise replacement after the RSA. The optimal intensity of stimulation
240 was determined by consecutive and incremental doublet pulses (100 Hz; Db100) to the
241 relaxed muscle until reaching the twitch force plateau (34). Supramaximal stimulation was
242 ensured by increasing the stimulation intensity by 20%. The femoral nerve electrical
243 stimulations were composed by a Db100 superimposed to MVC (Db100sup) and potentiated
244 Db100 on relaxed muscle 5 s after MVC termination (Figure 2).
245 Force signal analysis was carried out according to Milioni et al. (34) using specific MatLab
246 algorithms (The Math Works Inc, Natick, MA, USA). The voluntary activation (VA) was
247 calculated as VA = [1 - (Db100sup × (force level at stimulation / MVC) / Db100)] × 100 (34).
248 The intraclass correlations and coefficient of variations measured for MVC, Db100 and VA

249 were 0.82 (0.59 – 0.92) and 4.8%; 0.70 (0.39 – 0.87) and 5.7%; and 0.62 (0.25 – 0.83) and
250 3.2%.
251
252 Muscle biopsy and biomolecular measurements
253 Muscle biopsy
254 Participants underwent muscle biopsies at Pre and Post HIIT+supplementation 48 – 72 h
255 following the last physical test. Following local anaesthesia of the m. vastus lateralis using
256 2% lidocaine without vasoconstrictor, an incision of approximately 0.5-1.0 cm was made.
257 Muscle samples were obtained using a modified Bergstrom needle with suction; ~60-100 mg
258 fragments were immediately frozen in liquid nitrogen and stored at -80°C. Three participants
259 refused to participate in the Post HIIT+supplementation biopsy (one from Gβ and two from
260 GP), resulting in a final sample size for biomolecular measurements of Gβ: n = 8; and GP: n
261 = 6.
262 Chromatographic determination of muscle carnosine content
263 Muscle carnosine was determined by using HPLC (Hitachi, Hitachi Ltd., Tokyo, Japan), as
264 thoroughly described by Saunders et al. (41). Ten random samples were quantified in
265 duplicate to verify the coefficient of variation (coefficient of variation = 6.5%).
266
267 Western blot measurements
268 Approximately 20 mg of wet muscle was used as previously described by da Rocha et al.
269 (37,38). The antibodies used were PFK (Cell Signaling Technology, #8164s), MCT4
270 (Millipore Corporation, AB3316P), HIF-1α (Cell Signaling Technology, #14179s) and
271 glyceraldehyde 3-phosphate dehydrogenase (GAPDH – Cell Signaling, #2118s) (dilution

272 1:1,000). The transfer efficiency onto nitrocellulose membranes was confirmed by brief
273 staining of the blots with the Ponceau red stain. The protein/enzymatic content measured at
274 each time point (Pre and Post HIIT+supplementation) were normalized by the respective
275 standard protein content (i.e., GAPDH).
276
277 Muscle buffer capacity in vitro (βmin vitro)
278 Muscle buffer capacity was performed according to Bishop et al. (7). An aliquot of dry
279 muscle (~2.0 mg) was homogenized in a solution of sodium fluoride (NaF-10mM) (33 μL of
280 NaF.g-1 dm). This homogenate was maintained at 37°C (Thermomixer, Eppendorf, Germany)
281 and adjusted to a pH of 7.2 by the addition of NaOH (0.02 mM). The monitoring of pH was
282 performed by a glass microelectrode (Microelectrodes, Mettler Toledo, Switzerland)
283 connected to a pHmeter (FiveEasy Plus, Mettler Toledo, Switzerland). The homogenate was
284 then titrated by a sequence of additions of 2 μl HCl (10 mM) until a pH of 6.2 was reached.
285 All pH measurements were performed three times and the mean value used. From these data,
286 the number of moles of hydrochloric acid required to change the pH from 7.2 to 6.5 was
287 adjusted by the amount of dry muscle aliquoted in the sample for the determination of βmin
288 vitro.
289
290 Statistical analysis
291 Performance data were reported as mean ± SD while biomolecular data were reported as
292 mean ± standard error (SE). Homogeneity was confirmed by the Shapiro-Wilks test. A
293 general linear model was applied for TINC (time [Baseline × Pre × Post] × group [Gβ × GP]),
294 TSUPRA, RSA, biomolecular measurements (time [Pre × Post] × group [Gβ × GP]) and NMA
295 (condition [rest × fatigue] × time [Pre × Post] × group [Gβ × GP]) variables. Sidak’s post hoc

296 was used in case of significant F value. The percentage of change between Pre to Post
297 HIIT+supplementation (Δ%) of Tsupra, RSA, muscle carnosine content, βmin vitro, PFK,
298 MCT4 and HIF-1α of each group were analyzed using independent samples t test. Pearson’s
299 correlations were used to determine the relationship between the improvement of RSA
300 variables and absolute change of muscle carnosine content and βmin vitro. Significance level
301 was set at P ≤ 0.05. The analyses were performed using the statistical package SPSS 17.0 for
302 Windows.
303
304 RESULTS
305 HIIT efficacy
306 The HIIT led to significant improvement from baseline to Post HIIT+Supplementation (i.e.,
307 10 weeks) of Vሶ Omax (Gβ: +10.4 ± 2.2%; GP: +11.3 ± 6.9%) and maximal aerobic velocity
308 (Gβ: +11.4 ± 4.6%; GP: +11.4 ± 7.3%). However, only Gβ presented significantly increased
309 blood lactate concentration after TINC (Gβ: +29.0 ± 18.5%; GP: +9.9 ± 13.0%) (Figure 3).
310
312
313 Repeated sprints performance
314 Significant decrement of total time (P = 0.0001), best time (P = 0.002) and mean time (P =
315 0.0001) were observed only for Gβ. Also, only Gβ increased the RSA blood lactate
316 concentration after HIIT+Supplementation (P = 0.004). The Gβ worst time showed a
317 significant time effect (P = 0.02), although post hoc analysis comparing Pre- and Post
318 HIIT+supplementation did not reach statistical significance (P = 0.06). Fatigue index showed

319 no time effect (P = 0.34) and no time × group interaction was observed for any RSA variable
320 (all P > 0.09). The percentage of change of best time (P = 0.02) and peak blood lactate (P =
321 0.047) were significantly higher for Gβ compared to GP (Figure 4).
322
324
325 Muscle carnosine content and βmin vitro
326 There was a significant time effect (P = 0.001) for muscle carnosine content with significant
327 increases only for Gβ at Post HIIT+supplementation (P = 0.0001). There was a significant
328 time × group interaction (P = 0.001) with higher values of muscle carnosine content for Gβ
329 compared to GP at Post HIIT+supplementation (P = 0.003). The percentage of change
330 between Pre and Post HIIT+Supplementation of muscle carnosine content were greater for
331 Gβ compared to GP (P = 0.01).
332 There was no time x group interaction (P=0.87) for βmin vitro, although there was a significant
333 effect of time (P = 0.02) with greater overall values at Post HIIT+supplementation. However,
334 the percentage of change of βmin vitro between groups were not statistically different (Figure
335 5).
336
338
339 Correlations between absolute change of muscle carnosine content and βmin vitro with the
340 improvement of RSA variables

341 Significant associations were shown between muscle carnosine content and the improvement
342 of RSA total time (r = 0.62; P = 0.02) and mean time (r = 0.62; P = 0.02) (Supplemental Fig.
343 S1, https://figshare.com/s/dc44146b7a68750258de). The βmin vitro was not significantly
344 associated with any RSA variable (-0.17 < r < 0.11; P > 0.23).
345
346 Running performance at 115% of maximal aerobic velocity
347 Time to exhaustion (P = 0.33) and peak blood lactate concentration (P = 0.20) showed no
348 significant time effect after HIIT+supplementation. Oxygen uptake at exhaustion (P = 0.001)
349 was improved only for GP. There was no significant group × time interaction for any TSUPRA
350 variable measured (P > 0.08). The percentage of change of all TSUPRA variables did not
351 present significant differences (Figure 6).
352
354
355 Western blot measurements
356 The content of PFK, MCT4 and HIF-1α did not show any significant time (P > 0.17) or
357 interaction (P > 0.09) effects. However, the percentage change of HIF-1α was higher for Gβ
358 compared with GP (P = 0.03), while MCT4 also showed a trend towards greater increases
359 with Gβ (P = 0.08) (Figure 7).
360
362

363 Neuromuscular function
364 MVC (Gβ: 604 ± 96 N; GP: 630 ± 75 N; P = 0.54), Db100 (Gβ: 268 ± 37 N; GP: 294 ± 18 N;
365 P = 0.09), VA (Gβ: 88 ± 9%; GP: 88 ± 8%; P = 0.97) and jump height (Gβ: 38 ± 5 cm; GP:
366 37 ± 5 m; P = 0.64) were not different between Gβ and GP at Pre HIIT+supplementation.
367 MVC and jump height showed a significant condition effect (P = 0.0001) with significantly
368 lower values in the fatigued state compared to rest for both groups (P < 0.02) at both times
369 (Pre and Post HIIT+supplementation). There was no significant time (P = 0.66) or interaction
370 (P = 0.57) effect for either variable.
371 The Db100 showed a significant condition effect (P = 0.0001), with lower values in the
372 fatigued state compared to rest for GP at both times (Pre: P = 0.01; Post: P = 0.01) and for
373 Gβ only at Pre HIIT+supplementation (P = 0.02). A significant time effect was observed only
374 for GP (P = 0.01), with lower values in the fatigued state of Post HIIT+supplementation
375 compared to fatigued state at Pre HIIT+supplementation (P = 0.02). No significant
376 interaction (P = 0.28) was shown for Db100.
377 There was a significant condition effect (P = 0.003) for VA, with lower values in the fatigued
378 state compared to rest for GP at both times (Pre: P = 0.02; Post: P = 0.01) and for Gβ only at
379 Pre HIIT+supplementation (P = 0.02). There was a significant interaction (P = 0.04) with
380 higher values for Gβ in the fatigued state of Post HIIT+supplementation compared to GP (P =
381 0.02). A significant time effect was shown (P = 0.003) and Gβ presented significant higher
382 value at fatigued state at Post HIIT+supplementation compared to the fatigued state at Pre
383 HIIT+supplementation (P = 0.02), while GP presented significant lower values in the
384 fatigued state at Post HIIT+supplementation compared to fatigued state at Pre
385 HIIT+supplementation (P = 0.03) (Table 1).
386

387 ***** Table 1 near here*****
388
389 DISCUSSION
390 The aim of the present study was to investigate the influence of β-Alanine supplementation
391 during a HIIT program on RSA performance. Following the 6-week HIIT+supplementation
392 protocol, the Gβ group increased muscle carnosine content without a concomitant significant
393 increase in βmin vitro. Gβ, but not GP, improved some measures of RSA performance (i.e.,
394 total time, best time and mean time), and increased blood lactate concentration after the RSA
395 test. No significant alterations in supramaximal test performance or PFK content were shown;
396 however, percent increases in HIF-1α content for Gβ was significant compared to GP, while
397 MCT4 presented a trend (P = 0.08) for the same outcome. Finally, we observed a potential
398 neuromuscular protection of β-Alanine supplementation, as evidenced by lower central
399 neuromuscular deficit after the RSA. Collectively, this study demonstrated the potential of β-
400 Alanine supplementation as an ergogenic aid to individuals engaged in HIIT.
401 In the current study, the supplementation protocol was administered concomitantly to a HIIT
402 program aiming to reproduce the common practice of β-Alanine users. The RSA total time,
403 best time and mean time were reduced for Gβ, and the percentage change of Gβ best time
404 was higher, compared to GP, suggesting a positive influence of β-Alanine supplementation
405 on these markers of RSA performance. muscle carnosine content increased by ~85%, with no
406 change in GP (~1%), corroborating previous research showing an increase in muscle
407 carnosine content with β-Alanine supplementation (21,22,41), but not with HIIT (3). On the
408 other hand, a recent study did show that HIIT could increase muscle carnosine content in a
409 vegetarian population (39). While it remains disputable whether exercise training per se can
410 increase muscle carnosine content in omnivorous individuals, the noticeable β-Alanine–

411 induced increased muscle carnosine content may have contributed to the further improvement
412 of RSA performance shown, as there was a significant correlation between muscle carnosine
413 content and improvement of RSA. Since there were no similar improvements in RSA in the
414 placebo group, this suggests that the increases in muscle carnosine content may have been
415 responsible for these performance gains, and highlights the importance of muscle carnosine
416 content for high-intensity exercise performance.
417 Despite an increase in muscle carnosine content, βmin vitro was not changed in either group.
418 Harris et al. (21) and Hill et al. (22) estimated from Henderson-Hasselback equation that the
419 contribution of muscle carnosine content to muscle buffer capacity is approximately +9%,
420 which increases to ~14% after 4 weeks of β-Alanine supplementation with an increment of
421 ~64% in muscle carnosine content (21). Only Gross et al. (19) used the βmin vitro technique to
422 measure muscle buffering capacity after β-Alanine supplementation and similarly reported no
423 changes. This result is somewhat surprising in light of carnosine’s role as a muscle buffer.
424 Nonetheless, McGinley and Bishop (31) and De Salles Painelli et al. (39) recently questioned
425 the validity of the βmin vitro technique due to high inter-sample and intrasubject variability.
426 Indeed, in the current study there was a similar +15% increase in buffering capacity in the
427 placebo group; it is unclear as to whether this is due to the training stimulus or
428 methodological issues. The sample homogenization process may lead to an overestimation of
429 buffering capacity due to the inclusion of a pool of buffer substances from different
430 intracellular compartments, as well as extracellular proteins (31). Thus, in light of the
431 limitations inherent to this method, these data should be interpreted with caution.
432 The improved RSA performance could not be explained by alterations in supramaximal
433 running performance (TSUPRA outcomes), or any alterations in PFK content. This outcome
434 may be due to the HIIT protocol adopted in the present investigation, since recent evidence
435 suggests that this protocol only leads to discrete improvements in anaerobic capacity (36).

436 The current protocol was adopted due to its applicability to the real-world. Most HIIT
437 programs are extremely demanding, making them intolerable and unappealing to most
438 individuals (18). It is important to highlight, that an intense open-ended HIIT model
439 combined with β-Alanine supplementation loading prior to the HIIT block led to greater
440 improvements in RSA performance, although this was in trained individuals (4). Belllinger
441 and Minahan (4) recruited trained cyclists and administered 28 days of β-Alanine
442 supplementation loading (6.4 g.day-1) followed by 5 weeks of sprint interval training twice a
443 week (SIT - 4 × 1-km maximal cycling sprints with 4 min of active recovery) alongside a
444 maintenance dose of 1.2 g.day-1 of β-Alanine. The authors observed increased training
445 intensity throughout SIT as well as additional benefits during exhaustive extreme-intensity
446 cycling compared to placebo group (sprint interval training alone). Since our training
447 protocol clamped training intensity, it cannot be ruled out that further or different adaptations
448 may have occurred if the participants could increase their training intensity.
449 In the current study, only GP showed an increased oxygen uptake at exhaustion during the
450 TSUPRA with no other significant alterations. Conversely, Bellinger and Minihan (4) showed
451 only the group supplemented with β-Alanine improved anaerobic capacity, increased blood
452 lactate concentration and supramaximal cycling performance at 120% of Vሶ Omax . This
453 suggests that improvements in anaerobic capacity may be dependent upon training intensity
454 (4). On the other hand, similar to Bellinger and Minihan (4), we also showed an increased
455 blood lactate concentration in Gβ after RSA (and the incremental test) at Post
456 HIIT+supplementation.. Lactate has been suggested to be a powerful signaling molecule (16)
457 and has the potential to reciprocally control mechanisms with HIF-1α (35). HIF-1α is a key
458 transcription factor that, in addition to mediate adaptation of anaerobic glycolysis, can
459 upregulate the content of MCT4 (35,45). This is somewhat in line with the results of the
460 present investigation since we showed significant increases in blood lactate concentration, a

461 higher percentage change of HIF-1α and a trend towards the same result for MCT4 only for
462 Gβ.
463 The improvements in neuromuscular outcomes further support the hypothesis that increased
464 muscle carnosine content delayed muscle fatigue in this study. The RSA induced a significant
465 drop in all neuromuscular variables at Pre HIIT+supplementation, corroborating previous
466 literature which demonstrated that both peripheral and central fatigue contribute significantly
467 to the decline of RSA performance (25). However, after the intervention, only GP continued
468 to present significant peripheral and central neuromuscular deficits (drop of Db100 and VA)
469 after RSA, and Gβ presented higher values of VA after RSA than GP. The potential
470 contribution of a pH reduction during repeated sprints for neuromuscular fatigue remains
471 controversial (32), although there is evidence to suggest it occurs (17). It is possible to
472 speculate that the increased muscle carnosine content in Gβ may have prevented the negative
473 effects of hydrogen accumulation on contractile function at the cross bridge level (17), or the
474 disruption of the so-called “critical fatigue threshold”, theoretically responsible for
475 constraining central motor drive via feedback from group III/IV neural afferents (1),
476 contributing to the maintenance of Db100 and VA.
477 Muscle carnosine may also protect against neuromuscular fatigue due to changes in calcium
478 sensitivity. Dutka and Lamb (14)showed an interaction between carnosine and facilitation of
479 the muscle excitation-contraction process in isolated muscle fibres by increasing the
480 sensitivity of the muscle fiber to calcium ion, although Hannah et al. (20) did not confirm
481 these results in vivo. Further work should determine the mechanistic role of muscle carnosine
482 in calcium sensitivity.
483 An important limitation of the present study is the time-delay between the cessation of the
484 exercise and the MVC and peripheral electrical stimulation (~ 3.5 min) which may have led

485 to an underestimation of the actual state of neuromuscular fatigue induced by RSA, since
486 substantial recovery may occur within minutes (34). This might be the reason for the absence
487 of a significant group × time interaction, especially of peripheral fatigue variables. The
488 strengths of this study include the use of β-Alanine supplementation during a HIIT routine
489 mimicking a “real-world” condition where this supplement is commonly applied. Also, the
490 comprehensive assessments employed in this study allowed determining the role of β-Alanine
491 on physiological, performance and mechanistic outcomes. Limitations of the study involve
492 the relatively short-term follow-up period, the low sample size and the subjects’
493 characteristics (i.e., recreationally trained individuals not engaged in sports competitions).
494 Further studies should address some of these limitations.
495
496 CONCLUSION
497 β-Alanine supplementation throughout a HIIT program improved some measures of repeated
498 sprint performance. The mechanisms underlying these improvements are linked to an
499 increased muscle carnosine content although we showed no significant alteration in muscle
500 buffer capacity. The attenuation of neuromuscular fatigue, especially central fatigue, may
501 also have contributed to the improvement of repeated sprints performance. Overall, this study
502 provides evidence that β-Alanine supplementation may be a useful dietary intervention to
503 prevent fatigue in individuals undergoing HIIT.
504
505 ACKNOWLEDGMENTS
506 The authors wish to thanks all participant for their enthusiastic efforts.
507

508 GRANTS
509 Fabio Milioni received doctoral scholarship from São Paulo Research Foundation (FAPESP)
510 #2016/02683-6. Rodrigo Araújo Bonetti de Poli received master scholarship from São Paulo
511 Research Foundation (FAPESP) #2016/17836-2. Bryan Saunders received grant from São
512 Paulo Research Foundation (FAPESP) 2016/50438-0. Alessandro Zagatto received grants
513 from CNPq Process 307719/2016-2. The study was financed by São Paulo Research
514 Foundation (FAPESP) #2016/11076-6 and Coordenação de Aperfeiçoamento de Pessoal de
515 Nível Superior - Brasil (CAPES) - Finance Code 001.
516
517 DISCLOSURES
518 The authors have no conflicting interests to disclose in relation to this work.
519
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680
681
682
683

684 FIGURE LEGENDS
685 Figure 1. Schematic recruitment of participants and progression of each stage of the study.
686 Figure 2. Schematic chart of experimental design. TINC: Incremental running test. HIIT:
687 High-intensity interval training. TSUPRA: Supramaximal running test. RSA: Repeated sprints
688 test. NMA: Neuromuscular function assessment.
689 Figure 3. Incremental running test results. A: Maximal oxygen uptake. B: Maximal aerobic
690 velocity. C: Peak blood lactate. Gβ: n = 9; GP: n = 8.
691 Figure 4. Repeated sprints performance. A: Total time. B: Best time. C: Mean time. D: Worst
692 time. E: Fatigue index. F: Peak blood lactate. Gβ: n = 9; GP: n = 8.
693 Figure 5. A: Muscle carnosine content. B: Percentage of change of muscle carnosine content
694 between Pre and Post HIIT+Supplementation for Gβ and GP. C: Muscle buffer capacity in
695 vitro (βmin vitro). D: Percentage of change of βmin vitro between Pre and Post
696 HIIT+Supplementation for Gβ and GP. Gβ: n = 8; GP: n = 6.
697 Figure 6. Supramaximal running test performance. A: Time to exhaustion at 115% of
698 maximal aerobic velocity. B: Oxygen uptake at exhaustion. C: Peak blood lactate. Gβ: n = 9;
699 GP: n = 8.
700 Figure 7. Content normalized by GAPDH of A: Phosphofructokinase (PFK). B:
701 Monocarboxylate transporter 4 (MCT4). C: Hypoxia-inducible factor-1α (HIF-1α). Gβ: n = 8;
702 GP: n = 6.
703

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Table 1. Outcomes from neuromuscular function assessment normalized by their respective rest values at Pre HIIT+supplementation.
Gβ GP
Pre- Post- Pre- Post-
HIIT+Supplementation HIIT+Supplementation HIIT+Supplementation HIIT+Supplementation
Raw Rest Fatigued Δ% Rest Fatigued Δ% Raw Rest Fatigued Δ% Rest Fatigued Δ%
603,9± 93,0± -7,0± 102,4± 95,8± -6,4± 630,0± 88,2± -11,8± 97,6± 88,9± -8,9±
MVC 100 100
95,7 (N) 8,5* 8,5 10,32 9,7* 3,4 75,0 (N) 9,2* 9,2 10,8 12,8* 8,3
268,1± 95,1± -4,9± 93,4± 89,3± -4,0± 293,8± 94,0± -6,0± 93,6± 83,2± -11,4±
Db100 100 100
36,6 (N) 3,9* 3,9 12,2 12,9 10,2 18,3 (N) 6,9* 6,9 8,4 12,5*# 8,0
88,2± 94,2± -5,8± 100,6± 99,1± -1,2± 88,0± 94,3± -5,7± 98,4± 89,3± -9,2±
VA 100 100
8,5 (%) 7,7* 7,7 4,2 7,1# 8,9 8,4 (%) 4,4* 4,4 4,1 8,1*#$ 6,9
Jump 38,2± 87,4± -12,6± 104,6± 88,6± -15,0± 36,9± 86,2± -13,8± 100,1± 83,2± -16,7±
100 100
heigth 5,4 (cm) 5,9* 5,9 3,7 7,0* 9,3 5,0 (cm) 8,1* 8,1 5,7 7,2* 8,4
MVC: Maximal voluntary isometric contraction of knee extension; Db100: Amplitude of doublet 100 Hz; VA: Voluntary activation. *Significant different of
respective rest values (P < 0.03); #Significant different of fatigued condition at Pre-HIIT+Supplementation (P < 0.03); $ Significant different of Gβ in fatigued
condition at Post-HIIT+supplementation.

BETA ALANINA the Effect of Β-Alanine Supplementation During High-Intensity Interval Training on 3 Repeated Sprint Ability Performance and Neuromuscular Fatigue

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BETA ALANINA the Effect of Β-Alanine Supplementation During High-Intensity Interval Training on 3 Repeated Sprint Ability Performance and Neuromuscular Fatigue

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1 TITLE

2 The effect of β-alanine supplementation during high-intensity interval training on

3 repeated sprint ability performance and neuromuscular fatigue

5 RUNNING HEAD: β-Alanine during HIIT improves performance

10 Alessandro Moura Zagatto1,6*.

12 AFFILIATION & ADDRESSES

14 (UNESP), Laboratory of Physiology and Human Performance, Bauru, SP, Brazil

16 Rheumatology Division; Faculdade de Medicina FMUSP, University of São Paulo, Sao

17 Paulo, SP, Brazil

21 Paulo (USP), Ribeirão Preto, SP, Brazil

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23 University (UFMS), Campo Grande, MS, Brazil

25 Education, Bauru, SP, Brazil

26 *Corresponding author: São Paulo State University (UNESP), Faculty of Sciences,

27 Department of Physical Education, Laboratory of Physiology and Sport Performance.

30 E-mail: azagatto@yahoo.com.br Phone: +55 (14) 31039628

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33 The study investigated the influence of β-Alanine supplementation during a high-intensity

35 was randomized, double-blinded and placebo-controlled. Eighteen men performed an

40 HIIT+supplementation, participants performed the following tests: 1) TINC; 2) supramaximal

42 neuromuscular function was assessed by vertical jumps, maximal isometric voluntary

43 contractions of knee extension and neuromuscular electrical stimulations. Muscle biopsies

45 (βminvitro) and content of phosphofructokinase (PFK), monocarboxylate transporter 4 (MCT4)

49 time interaction was shown after HIIT+Supplementation for muscle carnosine

50 (Gβ:34.4±2.3mmol.kg-1dm and GP:20.7±3.0mmol.kg-1dm; p=0.003) and neuromuscular

52 or group-by-time interaction were shown for supramaximal running test performance,

55 contribute to an enhancement of RSA performance.

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58 sprint performance. The improvement of muscle carnosine content induced by beta-alanine

59 supplementation may have contributed to an attenuation of central fatigue during repeated

60 sprint. Overall, β-Alanine supplementation may be a useful dietary intervention to prevent

Downloaded from www.physiology.org/journal/jappl at Univ of Florida (128.227.024.141) on October 19, 2019.

64 (46) and is an important pre-cursor in muscle carnosine (β-alanyl-L-histidine) synthesis (40).

65 Muscle carnosine is a dipeptide that performs several physiological roles including

71 strategy to minimize muscle acidosis (23,40) induced by repeated all-out sprints.

76 phosphofructokinase (PFK - key regulatory glycolytic enzyme) activity is downregulated by

80 threshold of peripheral fatigue to be reached (1,25).

81 This impairment of anaerobic glycolytic activity (28,47,48) and subsequent neuromuscular

83 to increase muscle buffering capacity, the effect of β-Alanine supplementation on RSA

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91 carnosine would allow a greater effectiveness of anaerobic glycolysis, improving

92 performance and consequently generating a higher blood lactate concentration. Recent

96 expression of the monocarboxylate transporter 4 (MCT4) (35,45). Similarly, improved

97 capacity to handle intramuscular hydrogen accumulation would protect against

98 neuromuscular fatigue. However, there is no information regarding which mechanisms at the

99 biomolecular level would be modulated by β-Alanine supplementation, especially when

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118 neuromuscular fatigue; and 4) enhance RSA performance.

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138 ***** Figure 1 near here*****

140 Experimental design

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160 PFK, MCT4 and HIF-1α. (Figure 2).

162 ***** Figure 2 near here*****

164 Incremental running test (TINC)

174 High Intensity interval training (HIIT)

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183 to training program with 98% of all sessions completed.

185 Supplementation protocol

188 mg gastro-resistant capsules coated with hydroxypropyl-methylcellulose (DRcaps, Capsugel,

190 meal) to avoid paraesthesia (23,41).

193 remaining participants self-reported 100% of adherence to the supplementation protocol.

195 Supramaximal running test (TSUPRA)

138 * Figure 1 near here*

162 * Figure 2 near here*

311 * Figure 3 near here*

323 * Figure 4 near here*

337 * Figure 5 near here*

353 * Figure 6 near here*

361 * Figure 7 near here*