THE EFFECTS OF A PROPRIETARY MULTI-INGREDIENT ERGOGENIC SUPPLEMENT ON BODY COMPOSITION DURING HIGH-VOLUME PERIODIZED RESISTANCE TRAINING.

Transcription

1 THE EFFECTS OF A PROPRIETARY MULTI-INGREDIENT ERGOGENIC SUPPLEMENT ON BODY COMPOSITION DURING HIGH-VOLUME PERIODIZED RESISTANCE TRAINING A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Kinesiology By Dean Joseph Directo 2017

2 SIGNATURE PAGE THESIS: THE EFFECTS OF A PROPRIETARY MULTI- INGREDIENT ERGOGENIC SUPPLEMENT ON BODY COMPOSITION DURING HIGH- VOLUME PERIODIZED RESISTANCE TRAINING AUTHOR: Dean Joseph Directo DATE SUBMITTED: Spring 2017 Kinesiology and Health Promotion Department Dr. Edward Jo Thesis Committee Chair Kinesiology and Health Promotion Dr. Michael Liang Committee Member Kinesiology and Health Promotion Dr. Marcus Elam Committee Member Human Nutrition & Food Science ii

3 ABSTRACT In addition to resistance training methodology, nutrient intake remains a critical factor for the support of skeletal muscle metabolism, performance, and adaptation. Despite the traditional debate regarding the ideal type, timing, and quality of nutrients for recovery and/or performance, the consumption of exogenous substances like branchedchain amino acids (BCAA), beta-alanine, creatine, glutamine and various stimulant compounds (e.g. piperine) have previously shown to facilitate resistance training programs by supporting performance, recovery, and metabolic adaptations in skeletal muscle. This has largely enabled the use of multi-ingredient ergogenic supplements (MIES) that incorporate a single blend of these substances with the intent of obtaining a synergistic ergogenic effect. What remains relatively unclear is the efficacy by which a MIES that incorporates a blend of creatine, beta-alanine, glutamine, BCAA, and black pepper extract facilitates the adaptive changes in body composition during a high-volume resistance training regimen. Therefore, the purpose of this study is to investigate the effects of a proprietary MIES comprised of BCAA, beta-alanine, creatine, glutamine, and black pepper fruit extract (i.e. piperine) on body composition during 6 weeks of highvolume periodized resistance training. Thirty-two recreationally trained males (n=16) and females (n=16) were recruited to participate in this randomized, double blind, placebo controlled investigation. Subjects were assigned to either a placebo (PLA) (8 males and 8 females) or supplement group (EXP) (8 males and 8 females) in a randomized, counterbalanced manner. All subjects completed a 6 week periodized resistance training program 3 days a week with 48 hours of rest between each session. EXP and PLA consumed one serving of a proprietary MIES and placebo, respectively, immediately iii

4 post-workout and before sleep on training days, and two servings during rest days. Subjects underwent laboratory assessments for whole body composition via Dual Energy X-Ray Absorptiometry (DXA) and anthropometric measurements pre- and post-training. EXP (+1.7 ± 0.3 kg, p<0.001) and PLA (+1.5 ± 0.2 kg, p<0.001) demonstrated an increase in total body mass with no group x time interaction. EXP demonstrated a greater increase in lean mass than PLA (EXP= 2.1 ± 0.3 kg vs. PLA= 1.1 ± 0.3 kg) (p=0.03). There was a significant group x time interaction for FM (EXP= -0.4 ± 0.2 kg vs. PLA= +0.4 ± 0.2 kg) (p=0.02). EXP exhibited a decrease in body fat percentage (1.2 ± 0.3 % units) (p=0.01) while PLA showed no change. A group x time interaction (p=0.01) revealed that post-training body fat percentage was lower in EXP than PLA. There were no between-group differences for average daily training volume each week. Supplementation of a proprietary MIES comprised of branched-chain amino acids, betaalanine, creatine hydrochloride, glutamine, and black pepper fruit extract may enhance body composition changes during high-volume periodized resistance training. iv

5 TABLE OF CONTENTS Signature Page... ii Abstract... iii List of Figures... vi List of Tables... vii Chapter 1: Introduction... 1 Statement of the Problem... 2 Purpose Statement... 2 Significance of the Study... 2 Specific Aims... 3 Hypothesis... 3 Limitations... 3 Delimitations... 3 Operational Definitions... 4 Chapter 2: Literature Review... 5 Dietary Ergogenic Aids for Resistance Training Adaptations... 7 Creatine... 7 Beta-Alanine... 9 Branched Chain Amino Acids Glutamine Black Pepper Extract (Piperine) Evidence for Multi-Ingredient Ergogenic Supplements...15 v

6 Future Research Implications and Conclusions...16 Chapter 3: Methodology Experimental Design Subjects...19 High-Volume Periodized Resistance Training Protocol...20 Dietary Supplementation Protocol Laboratory Testing Procedures Maximal Muscular Strength Body Composition Statistical Analyses Chapter 4: Results...25 Descriptive Measures...25 Body Composition Measures...25 Total Body Mass...25 Lean Mass...26 Fat Mass...27 Body Fat Percentage...28 Dietary Intake...29 Training Volume...32 Chapter 5: Discussion...33 References vi

7 LIST OF FIGURES Figure 1 Schematic of Experimental Timeline...19 Figure 2 Pre- to post-training change in Total Body Mass for EXP and PLA...26 Figure 3 Pre- to post-training change in Lean Mass for EXP and PLA...27 Figure 4 Pre- to post-training change in Fat Mass for EXP and PLA...28 Figure 5 Pre- to post-training change in body fat percentage for EXP and PLA...29 Figure 6 Average daily caloric intake for each experimental week and total average caloric intake for EXP and PLA...30 Figure 7 Average daily protein intake for each experimental week and total average protein intake for EXP and PLA Figure 8 Average daily carbohydrate intake for each experimental week and total average carbohydrate intake for EXP and PLA...31 Figure 9 Average daily fat intake for each experimental week and total average fat intake for EXP and PLA...31 Figure 10 Weekly and average total volume between EXP and PLA...32 vi

8 LIST OF TABLES Table 1. Resistance Training Protocol Table 2. Between-group comparison of descriptive baseline measures vii

9 CHAPTER ONE Introduction Substantial evidence supports the use of periodized resistance training (RT) to achieve positive physiological adaptations in skeletal muscle and healthy body composition (Botero et al., 2013; Kraemer et al., 2000; Simão et al., 2012). Specifically, RT is arguably the most practical approach to experience the continuous and appropriate stress necessary to stimulate muscular growth and improvement of lean body mass (LBM) and physical function. To facilitate such adaptations, performance optimization during an exercise bout is an important factor to consider. Besides methods like proper nutrition, rest, and recovery strategies, dietary supplements have been a popular yet controversial approach to enhance RT performance and thereby, efforts to achieve muscular development. Exogenous dietary substances like branched-chain amino acids (BCAA), creatine, beta-alanine, glutamine, and various plant-based compounds have individually demonstrated efficacy in supporting RT performance and adaptation (Kreipke et al., 2015; Ormsbee et al., 2012; Ormsbee et al., 2013). Recently, the dietary industry has turned to the development of multi-ingredient ergogenic supplements (MIES) that incorporate a mixture of these ingredients in efforts to combine each of their unique properties into one product. However, there is currently limited evidence supporting a MIES comprised of a mixture of the aforementioned ingredients on RTinduced adaptations in skeletal muscle and overall body composition. Therefore, the purpose of this study is to examine the effects of a proprietary MIES incorporating BCAA, creatine, beta-alanine, glutamine, and black pepper fruit extract (piperine) on body composition during high-volume periodized resistance training. 1

10 Statement of the Problem Periodized resistance training has demonstrated substantial efficacy in generating positive adaptations in skeletal muscle that result in improved size, mass, function, and metabolism (Rhea, Ball, Phillips, & Burkett, 2002). The use of multi-ingredient ergogenic supplements (MIES) have shown some variable degree of efficacy, depending on dosing and ingredients, to support or facilitate these physiological adaptations primarily through nutrient and bioenergetics support for skeletal muscle. Multi-ingredient ergogenic supplements typically incorporate a mixture of empirically supported ergogenic substances such as BCAA, glutamine, beta-alanine, creatine, and black pepper fruit extract that purportedly enhance the quality of subsequent exercise performance and may lead to an increased rate of training adaptations. What remains relatively uncertain is the degree of efficacy by which intersession recovery consumption of an MIES comprised of the aforesaid ingredients enhances overall resistance training induced alterations to body composition. Purpose Statement The purpose of this study was to investigate the effects of a proprietary multiingredient ergogenic supplement comprised of branched chain amino acids, beta-alanine, creatine hydrochloride, glutamine, and black pepper fruit extract on body composition during 6 weeks of high-volume periodized resistance training. Significance of the Study The results of the proposed study are anticipated to provide further practical information regarding the application of multi-ingredient ergogenic supplements (MIES) in exercise training programs. Empirical information from the proposed investigation 2

11 may add insight to the systematic procedure for the effectiveness of intersession MIES consumption and training adaptations. Additionally, the proposed study, to our knowledge, will be one of the first to test the effects of a MIES composed specifically of a blend of BCAA, beta-alanine, creatine, black pepper fruit extract, and glutamine on RTinduced changes in body composition. Specific Aim In comparison to a placebo control, this study aimed to investigate the degree by which a MIES composed of BCAA, beta-alanine, creatine, black pepper fruit extract, and glutamine alters the changes in body composition during 6 weeks of high-volume periodized resistance training. Hypothesis In comparison to a placebo control, consumption of the experimental MIES will induce greater improvements in lean body mass and overall body composition following 6 weeks of high-volume periodized resistance training. Limitations The limitations to the proposed study included the following: 1) previous exercise training experience may have influenced the degree of adaptation during the resistance training protocol implemented during the present investigation; 2) dietary intake across the study period were self-recorded by subjects, and 3) some periods of supplementation were unsupervised. Delimitations The measure of analysis was delimited to healthy, recreationally trained male and female subjects between the ages of years. Participation were denied if the subject 3

12 had any major chronic diseases or physical conditions in which exercise cannot be continued during the length of the study. Participants who have allergies to milk and nut products were denied entry to the study. Operational Definitions One-Repetition Maximum (1RM) - The greatest load that can be moved through the entire range of motion properly for no more than 1 complete repetition for any given exercise. Six-Repetition Maximum (6RM) The greatest load that can be moved through the entire range of motion properly for no more than 6 complete repetitions for any given exercise Repetitions in Reserve - The estimated number of successfully completed repetitions following a bout of exercise. Resistance Exercise- Any activity that requires an acute increase in motor unit activation to generate a muscular force sufficient enough to overcome an external load imposed on the active muscle groups. Resistance Training- A long term exercise program consisting of repeated bouts of resistance exercise with the intent of improving or maintaining muscular strength. Neural Adaptations- Change over time in the responsiveness of the sensory system to a constant stimulus. 4

13 CHAPTER TWO Literature Review There is a substantial body of evidence demonstrating the efficacy by which RT confers the appropriate stressors needed for hypertrophic adaptations in skeletal muscle. (Spiering et al., 2008). It is well understood that events at the molecular level compose changes at the cellular level, and exercise physiology research has uncovered some of the molecular mechanisms responsible for skeletal muscle hypertrophy and growth in response to RT. The continual overload stress applied to the skeletal muscle system during RT provides an appropriate stimulus for intracellular anabolic signaling pathways that regulate muscle protein synthesis (MPS) (Bodine et al., 2001; Hornberger et al., 2004). These anabolic signals in skeletal muscle protein metabolism over time result in cellular hypertrophy and eventually whole tissue growth (Spiering et al., 2008). Ultimately, one may experience noticeable improvements in lean body mass (LBM) and overall body composition. Increased awareness for optimum body composition has largely enabled the practice of RT and also, the concurrent use of dietary supplements to support adaptations. The term ergogenic aids (or ergogenic supplements) has been previously used to describe exogenous substances that are consumed with the intent of enhancing physical performance and/or supporting any physiological adaptations to training such as skeletal muscle growth. In fact, the use of ergogenic aids, including multi-ingredient ergogenic supplements (MIES), has increased by approximately 64% in young athletes since 2010 (Hoyte, Albert, & Heard, 2013). Given that RT has shown through substantial evidence to promote positive muscular adaptations it has become common practice to explore 5

14 various dietary strategies, like ergogenic supplements, to further optimize progress towards improved body composition (Maughan, Depiesse, Geyer, & Federations, 2007; Young & Stephens, 2009). In the context of skeletal muscle mass development, the intent of incorporating ergogenic supplementation into RT programs remains two-fold: 1) to acutely enhance performance and thereby muscular work during individual training bouts, and 2) to provide nutritional support for skeletal muscle recovery and growth (Kreipke et al., 2015; Naderi, Earnest, Lowery, Wilson, & Willems, 2016; Ormsbee et al., 2012). A multitude of exogenous substances have been previously studied yet a small percentage of these supplements have shown ergogenic effects (Outram & Stewart, 2015). For instance, creatine, beta-alanine, and certain plant-based bioactive compounds have demonstrated efficacy in aiding RT performance by mitigating fatigue or supporting bioenergetic mechanisms (Cooper, Naclerio, Allgrove, & Jimenez, 2012; Culbertson, Kreider, Greenwood, & Cooke, 2010; Hill et al., 2007; Kreipke et al., 2015). Other supplements, like branch chain amino acids (BCAA) and other essential amino acids have shown to facilitate skeletal muscle recovery and provide nutrient provision for MPS and thus, myocellular hypertrophy (Kreipke et al., 2015; Ormsbee et al., 2012; Ormsbee et al., 2013). Each of the aforesaid substances have largely been studied individually in terms of their effects on various indices of performance as well as body composition changes following a RT protocol. However, less is known regarding the efficacy by which a MIES incorporating a combination of these substances supports or enhances the adaptive response to RT. The MIES industry has grown tremendously based on the notion that a blend of supported bioactive ingredients may be most efficient and optimum for ergogenic effects. However, the production and use of the MIES approach fails to 6

15 accompany a concomitant body of empirical support for their proposed effects. Thus, it may be reasonable to suggest that the MIES strategy remains largely unexplored with no substantial knowledge as to the optimum combination of ingredients in both type and dosage. Dietary Ergogenic Aids for Resistance Training Adaptations In order to facilitate the augmentation of performance and body composition alterations during RT, it may be of best interest for dietary supplement producers to identify exogenous substances that are empirically supported to possess ergogenic properties. To date, supplemental creatine, beta-alanine, BCAA, glutamine, and piperine have individually demonstrated some degree of efficacy in further promoting RT-induced adaptations (Cooper et al., 2012; Kreipke et al., 2015; Rawson & Volek, 2003; Sharp & Pearson, 2010). Previous research highlights mechanistic to whole-body level evidence for each of the aforementioned substances. Uncovering the interaction between these individual substances and exercise physiology may facilitate understanding of the potential advantages of a MIES incorporating a mixture of such ingredients. Creatine Creatine is a nitrogenous inorganic acid that can be found exogenously in protein rich foods, such as beef and fish (Harris, Söderlund, & Hultman, 1992). Creatine is also produced in vivo at approximately 1g per day collectively by the liver, kidneys and the pancreas (Cooper et al., 2012). Upon ingestion, digestion, and absorption, circulating creatine may be transported into various cells by creatine transporter, also referred to as CreaT1 (Cooper et al., 2012). Approximately 95% of total body creatine is found within skeletal muscle alluding to its critical role in muscle metabolism, in particular, 7

16 bioenergetics (Cooper et al., 2012). Specifically, creatine largely functions as a powerful energy substrate in its phosphorylated form, i.e. phosphocreatine (PCr) (P. Balsom, Ekblom, Söerlund, Sjödln, & Hultman, 1993). Phosphocreatine plays a pivotal role in human muscle bioenergetics particularly in the phosphagen pathway. In a single reaction catalyzed by creatine kinase, PCr is used to rephosphorylate adenosine diphosphate (ADP) into adenosine triphosphate (ATP), the cells energy currency (P. D. Balsom, Söderlund, & Ekblom, 1994; Volek et al., 1997). During exercise, especially high intensity exercise requiring a maximal rate of ATP turnover, the phosphagen system becomes the predominate bioenergetic system since its production of ATP is fastest among all available pathways (Ellington, 1989). However, this system is limited since PCr, the energy substrate, depletes proportional to the intensity of exercise (P. Balsom et al., 1993; Harris et al., 1992; Volek et al., 1997). Once PCr levels are depleted, maximal effort work cannot be sustained in skeletal muscle. Thus, maximizing resting muscle PCr levels would conceivably improve bioenergetics capacity especially during maximal effort exercise. The idea of exogenous creatine supplementation as an ergogenic aid is predicated on this contention. In support, Harris et al. (1992) reported a 20-50% increase in intramuscular PCr levels following multiple days of creatine monohydrate consumption of approximately g/day (Harris et al., 1992). Balsom et al. (1993) subsequently demonstrated that an increase in total intramuscular creatine delays the onset of fatigue during repeated bouts of high-intensity exercise (P. Balsom et al., 1993). Thus in terms of RT, creatine supplementation may enhance the overall performance of each bout, which would likely facilitate long-term muscular adaptations. 8

17 A large body of previous literature demonstrates the long-term effects of creatine supplementation on RT-induced adaptations in skeletal muscle (Brose, Parise, & Tarnopolsky, 2003; Burke et al., 2008; Cooper et al., 2012; Volek et al., 1999). For instance, Volek et al. (1999) showed that 12 weeks of concurrent creatine supplementation and RT enhanced fat-free mass (FFM), physical performance and muscle morphology. Specifically, creatine supplementation improved FFM by 6.3% while the placebo yielded only a 3.1% increase following 12 weeks of progressive RT (Volek et al., 1999). Corresponding to these whole-body outcomes, on the myocellular level, creatine supplementation compared to placebo demonstrated a significantly greater increase in type I (35% vs. 11%), type IIa (36% vs 15%) and type IIab (35% vs 6%) muscle fiber cross-sectional areas. Brose et al. (2003) subsequently confirmed these outcomes in a 14 week intervention further supporting the ergogenic value of exogenous creatine for RT-induced muscular growth (Brose et al., 2003). Based on the current evidence, as those presented above, the benefits of creatine supplementation are raising intramuscular PCr and supporting bioenergetics, thereby acutely enhancing high-intensity performance. In relation to RT, increasing the muscular work and therefore quality of each bout may result in greater improvements of muscle mass. Beta-Alanine Beta-alanine (BA) is a nonessential amino acid that is the rate-limiting precursor of the dipeptide, carnosine (Culbertson et al., 2010; Harris et al., 2006; Hill et al., 2007). Beta-alanine combines with L-histidine with the aid of carnosine synthase to form carnosine (Artioli, Gualano, Smith, Stout, & Lancha, 2010). Carnosine plays an important role in stabilizing intramuscular ph levels during moderate to high-intensity 9

18 activities. With increasing exercise intensity, acute metabolic acidosis may result from rapid accumulation of H+ ions from lactic acid and ATP hydrolysis (Maclaren, Gibson, Parry-Billings, & Edwards, 1989). This physiological state, often referred to as acute metabolic acidosis, is conducive to fatigue and limits optimum performance. The contention behind beta-alanine supplementation is to increase intramuscular carnosine levels and therefore the buffering capacity of active skeletal muscles during exercise. Dunnett et al. (1999) in a pioneer study, indicated that intramuscular carnosine levels were directly influenced by beta-alanine bioavailability as opposed to histidine (Dunnett & Harris, 1999). Subsequently, Harris et al. (2006) showed that supplementing 3 to 6 g of beta-alanine may enhance muscle carnosine concentrations (Harris et al., 2006). These findings were corroborated later by Derave et al. (2007) who demonstrated a significant increase in carnosine levels in the calf muscles of male sprinters after four weeks of beta-alanine supplementation of 4.8 g per day. As evidenced above, betaalanine supplementation is an effective strategy to increase intramuscular carnosine levels which may ultimately facilitate exercise performance. Subsequent investigations demonstrated the efficacy by which beta-alanine supplementation enhances various indices of exercise performance. For instance, Hill et al. (2007) found that beta-alanine supplementation raised intramuscular carnosine by 58.8% in 4 weeks and 80.1% in 10 weeks which accompanied a significant enhancement in high intensity cycling capacity (Hill et al., 2007). Accordingly, beta-alanine supplementation appears to also increase the muscular power output obtained at lactate threshold possibly due to enhanced buffering capacity of acidic H+ ions (Zoeller, Stout, O'kroy, Torok, & Mielke, 2007). Although multiple investigations support the acute 10

19 ergogenic effects of beta-alanine, there is a limited pool of data regarding its long-term effects on RT-induced muscular adaptations. However, based on the current level of evidence, RT performance may be likely enhanced in response to beta-alanine consumption due to its positive effects on buffering capacity and fatigue mitigation. Ultimately, these effects over time may result in superior adaptations in skeletal muscle such as muscular growth (Kern & Robinson, 2011; Smith et al., 2009; Walter, Smith, Kendall, Stout, & Cramer, 2010). Branched Chain Amino Acids Branched Chain Amino Acids (BCAA) are comprised of 3 essential amino acids; leucine, isoleucine and valine. BCAAs, especially leucine, play an important role in mediating the rate of protein synthesis and the rate of protein degradation in skeletal muscle (Blomstrand, Eliasson, Karlsson, & Köhnke, 2006). The proposed benefits of BCAA supplementation in the context of human performance and muscular development are increased protein anabolism, improved regeneration and recovery following exercise induced muscle damage, and bioenergetics support (Garlick, 2005; Kimball & Jefferson, 2006; Shimomura et al., 2010; Shimomura, Murakami, Nakai, Nagasaki, & Harris, 2004; Shimomura et al., 2006). In relation to improving skeletal muscle mass, BCAA supplementation may support underlying hypertrophic mechanisms, namely myocellular protein synthesis (Atherton, Smith, Etheridge, Rankin, & Rennie, 2010; Li, Yin, Tan, Kong, & Wu, 2011; Shimomura et al., 2004). Thus, BCAA supplementation is conducive to mediating overall protein balance and its effect on skeletal muscle metabolism. The ergogenic properties of BCAA supplementation is due to leucine s role in the activation of MPS. Leucine facilitates the activation of the mammalian target of 11

20 rapamycin (mtor) pathway through the phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) and p70 ribosomal s6 kinase (p70s6k1) (Atherton et al., 2010; Blomstrand et al., 2006; Kimball & Jefferson, 2006; Li et al., 2011). Once the mtor pathway and its downstream targets are activated, an increase in muscle protein synthesis follows (Atherton et al., 2010; Blomstrand et al., 2006; Kimball & Jefferson, 2006; Li et al., 2011). Atherton et al. (2010) investigated the distinct anabolic signaling response to each essential amino acids (EAA) in skeletal muscle cells. They concluded that the phosphorylation of mtor, 4E binding protein-1 (4EBP1), and ribosomal s6 kinase (p70s6k1) were increased in response to leucine stimulation compared to any other EAA (Atherton et al., 2010). In understanding leucine s direct phosphorylation of the mtor pathway, it may be beneficial to investigate the effects of BCAA supplementation with RT. Branched chain amino acid supplementation, in regards to RT, has shown to increase MPS (Howatson et al., 2012; Shimomura et al., 2010; Shimomura et al., 2006). Blomstrand et al. (2006) demonstrated significantly greater increases in the phosphorylation of mtor pathway 1 hour after exercise during oral BCAA administration (Blomstrand et al., 2006). Additionally, significantly greater increases in the phosphorylation in p70s6k1 were displayed up to 2 hours after RT compared to no oral administration of BCAAs (Blomstrand et al., 2006). Similarly, Shimomura et al. (2010) demonstrated elevated plasma BCAA concentrations 2hr post-exercise (Shimomura et al., 2010). Thus, orally administered BCAA may facilitate increased MPS following RT. 12

21 BCAA supplementation may also facilitate recovery by decreasing the severity of exercise-induced muscle damage. Shimomura et al. (2010) investigated the effects of BCAA supplementation on delayed-onset muscle soreness (DOMS) following exercise. They determined that soreness was significantly lower in the BCAA group post-exercise (Shimomura et al., 2010). Additionally, BCAA supplementation suppressed the decrease in muscle force during maximal voluntary isometric contractions (Shimomura et al., 2010). Howatson et al. (2012) investigated the effects of exercise-induced muscle damage following 100 consecutive drop-jumps and determined that exogenous BCAAs supplementation reduced the negative effects of damaging exercise by attenuating creatine kinase (CK) efflux (an effective biomarker for muscle damage in the blood), reducing residual muscle soreness and improving recovery of muscle function greater than a placebo control (Brancaccio, Lippi, & Maffulli, 2010; Howatson et al., 2012). As evidenced above, BCAA supplementation reduced indices of muscle damage and accelerated recovery. This may be due to BCAAs effect in improving MPS and thereby the extent of secondary muscle damage following exercise. Therefore, the benefits of BCAA supplementation are to increase protein anabolism, improve regeneration, and to decrease the severity of exercise-induced muscle damage. Glutamine Glutamine is a non-essential amino acid that is abundant in the body and makes up 60% of the free amino acid pool (Antonio, Sanders, Kalman, Woodgate, & Street, 2002; Rowbottom, Keast, & Morton, 1996). Glutamine plays an essential role in promoting and maintaining functions of various organs and cells, for example, the kidney, liver and heart (Curi et al., 2005). In the context of exercise, Glutamine is 13

22 responsible for preventing immunodeficiency responses following high-intensity exercise (Budgett, 1998; Lehmkuhl et al., 2003). As prolonged exercise continues, plasma concentrations of glutamine decrease leading to immunosuppression characteristics seen similarly with over-training (Neu, DeMarco, & Li, 2002; Petibois, Cazorla, Poortmans, & Déléris, 2002). Thus, the benefits of glutamine supplementation are to decrease the impairment of immunity functions following prolonged exercise. Castell et al. (1996) determined that glutamine supplementation consumed immediately after a marathon reduced the incidence of upper respiratory tract infections in the 7 days following the race (Budgett, 1998; Castell, Poortmans, & Newsholme, 1996; Neu et al., 2002). When supplementing glutamine with RT, Antonio et al (2002) investigated the effects of highdose glutamine supplementation on RT and showed no increase in weightlifting performance (Antonio et al., 2002). Although, glutamine supplementation may not directly enhance RT performance, the benefits of glutamine supplementation are to prevent immunosuppressive characteristics. Black Pepper Fruit Extract (Piperine) Black pepper extract (Piperine) is a naturally occurring compound present in various parts of plants from the family Piperaceae (V. Badmaev, Majeed, & Prakash, 2000). Piperine s main role is to increase the bioavailability of co-ingested constituents (Atal, Zutshi, & Rao, 1981). Although, there is not a definitive explanation to piperine s mechanisms of action, the ergogenic properties of piperine are hypothesized to work through its digestive passage in the liver (Ahmad et al., 2012; Atal et al., 1981; Khajuria, Thusu, & Zutshi, 2002; Srinivasan, 2007). Atal et al. (1981) demonstrated the effects of the trikatu group of acrids and the efficacy of bioavailability of certain drugs (long 14

23 pepper, black pepper and ginger), and determined that long pepper, black pepper and ginger had the capacity to significantly increase the bioavailability of certain drugs (Atal et al., 1981). The study hypothesized that the increase in bioavailability of the piperine is due to either/or; rapid absorption from the gastrointestinal tract, protection from being metabolized/oxidized in their first passage through the liver, or a combination of the two mechanisms (Atal et al., 1981). Piperine s potential to enhance bioavailability has also been investigated in drugs such as coenzyme Q10 (V. Badmaev et al., 2000), vasicine (Atal et al., 1981), pyrazinamide (U. Zutshi, 1989), rifampicin (R. K. Zutshi, Singh, Zutshi, Johri, & Atal, 1985), isoniazid (U. Zutshi, 1989), propranolol (Bano et al., 1991), theophylline (Bano et al., 1991), phenytoin (Bano, Amla, Raina, Zutshi, & Chopra, 1987), fat soluble beta-carotene (Vladimir Badmaev, Majeed, & Norkus, 1999), water soluble vitamins B6 and vitamin C (Majeed, Badmaev, & Rajendran, 1996). These findings are vital, as it provides insight into piperine s potential to enhance serum levels of drugs in humans. The ergogenic benefits of piperine may be due to its mechanisms of action regarding bioavailability when combined with other constituents. Thus, the benefits of black pepper extract supplementation are to increase the absorption and bioavailability of co-ingested ergogenic supplements. Evidence for Multi-Ingredient Ergogenic Supplements The ergogenic properties of ingredients such as; creatine, beta alanine, BCAA, glutamine and piperine, have shown through research to be ergogenic in isolation. When combined into a mixture of ingredients as a MIES, the research is limited, but the efficacy of MIES comprised of other mixtures shows promise. For instance, Ormsbee et al. (2012) conducted a six-week study that investigated the effects of pre-exercise and 15

24 post-exercise consumption of a MIES on cardiovascular health and body fat in trained men (Ormsbee et al., 2012). The MIPS, or multi-ingredient performance supplements termed by Ormsbee was comprised of; whey protein, casein, BCAAs, creatine, beta alanine and caffeine. The study determined that only peek anaerobic power was significantly improved in the MIES intervention group. Similarly, Kreipke et al. (2015) investigated the efficacy of a MIES on muscular strength, body composition, and anabolic hormones in RT young men for four weeks. The MIES utilized was comprised of beta alanine, BCAAs, long jack root extract, resveratrol, and black pepper extract (piperine). Utilizing these ingredients, the MIES group increased strength in the bench press, increased total weight lifted, and increased deadlift strength relative to total body mass and lean mass (Kreipke et al., 2015). Although the results from the two studies show ergogenic benefits and improvements in strength, power, and body composition, MIES with similar ingredients should be investigated under similar RT procedures to further investigate optimal ingredients. Future Research Implications and Conclusion Future research would benefit in the investigation of optimization of ingredients in an MIES. The current body of research regarding MIES would support that MIES demonstrate ergogenic effects, but to an unknown degree. Similar studies investigating MIES conducted by Kreipke et al. (2015) investigated the effects of an MIES with ingredients such as long jack root, BCAA, beta alanine, and other proprietary blends during 4 weeks of RT in trained males. The study determined that those that took the MIES saw significantly greater increases in bench press, total weight lifted, deadlift strength relative to total body mass, and significant increase in lean mass (Kreipke et al., 16

25 2015). Ormsebee et al (2012) also investigated a MIES blend of whey protein, casein protein, BCAAs, creatine, beta alanine, and caffeine during 6 weeks of RT on trained males and determined that the MIES intervention saw increases in peak anaerobic power compared to control interventions. Based off these two current studies that investigated the efficacy of MIES consumption, there is limited information regarding the optimum ingredients utilized in a MIES. As mentioned, future research should standardize ingredients and investigations should involve trained and untrained male and female individuals for a greater understanding of MIES. 17

26 CHAPTER THREE Methodology Experimental Design We implemented a randomized, counter-balanced, placebo-controlled experiment for the proposed study. Subjects were pair-matched by total body mass (TBM), lean body mass (LBM) and sex and placed into one of two groups for 7 weeks. The two groups were the experimental MIES group (EXP, n=15) and placebo group (PLA, n=24). However, due to subject dropout, the placebo group had 24 subjects while the MIES group had 15 subjects. EXP consumed an experimental MIES comprised of a blend of BCAA, beta-alanine, creatine hydrochloride, black pepper fruit extract, and glutamine while PLA consumed an isocaloric and taste-matched placebo. Each subject underwent a 6-week periodized resistance training program 3 days per week (Monday, Wednesday, and Friday) with 48 hours of rest between each session (experimental weeks 1-6) (Figure 2). Each group consumed 1 serving of their respective supplement (MIES or placebo) immediately post-workout, 1 serving before sleep on training days, and 1.5 servings during intersession rest days. Subjects visited the Human Performance Research Laboratory (HPRL) at California State Polytechnic University, Pomona (43-202b) on week-0 (pre-training) and week-7 (post-training) to complete testing procedures for body composition (DXA) and anthropometric measurements. 18

27 Figure 1. Schematic of Experimental Timeline. Subjects Healthy college-aged (18-32 years old), recreationally-trained male (n=19) and female (n=20) subjects were recruited for the study. Each subject completed a health and exercise history questionnaire and signed an informed consent form prior to participation. In order to participate in the study, the subjects must have met the following inclusion criteria: 1) years old of age, 2) not a competitive athlete in a sanctioned collegiate or professional sport, and 3) recreationally resistance-trained (i.e. resistance exercise is performed at least 3 days/week for 6 months prior to the start of the study and perform the back squat and bench press with a load 1.0 (male) or 0.75 (female) and 0.75 (male) or 0.5 (female) times their bodyweight, respectively). Subjects were excluded from participation if they reported a medical or surgical history that contraindicated the experimental protocol and/or confounded the interpretation of results. These included, but are not limited to: 1) cardiovascular, pulmonary, metabolic, or renal diseases, 2) 19

28 hypertension, 3) smoking, 4) use of any medication, including those with cardiovascular, pulmonary, hyperlipidemic, hypoglycemic, or hypertensive effects, and 5) milk allergies. In addition, subjects were excluded if they consumed dietary ergogenic aids daily within 6 weeks prior to the study. Daily use of nutritive supplements (e.g., whey protein or multivitamins) did not call for exclusion. Subjects were asked to maintain their normal dietary intake as it was monitored via an online-based dietary tracker. Subjects were also asked to refrain from other exercise training programs for the duration of the study. High-Volume Periodized Resistance Training Protocol (RT) The RT protocol was formatted based on the Daily Undulating Periodization training model (Zourdos et al., 2016) and comprised of three training sessions per week (Monday, Wednesday, and Friday) separated by 48 hours of rest between each session (Table 1). The RT protocol duration spanned 6 weeks (experimental weeks 1-6); with an addition of 2 familiarization sessions that were implemented in week 0 (lead-in week). During the RT period, Session 1 (Monday) incorporated the following exercises/lifts in the listed order: 1) back squat, 2) bench press, 3) deadlifts, 4) shoulder press, 5) rows, and 6) dumbbell chest press. Exercises 1-3 were performed for 4 sets for 12 repetitions at 60% 1 repetition maximum (RM), while the remaining exercises were performed for 3 sets at 10 repetitions with 10RM loads. Session 2 (Wednesday) incorporated the same exercises as session 1 but excluded the deadlifts. The back squat and bench press were performed for 4 sets for 10 repetitions at 65% 1RM while the remaining exercises were performed for 3 sets for 8 repetitions with 8RM loads. Session 3 (Friday) consisted of the same exercises as session 1 in the same order. The back squat, bench press, and deadlift 20

29 were prescribed at 5 sets for 8 repetitions at 70% 1RM while remaining exercises were prescribed at 3 sets for 6 repetitions with 6RM loads. Training load progression was made on a set-to-set basis and was determined by the subject s estimated repetitions in reserve (RIR) or the number of repetitions achieved within a set. For any given set for a given exercise, if a subject successfully completes the prescribed repetitions and estimates that up to 2 additional repetitions could have been performed (i.e. 2 RIR), no changes to the load were implemented during the subsequent set. Also, if the subject falls up to 2 repetitions short of the prescribed repetitions, no changes to the load were made for the subsequent set. If the subject successfully completed the prescribed repetitions and reports a RIR of 3-5, then 5 lbs. was added to the subsequent set. If the subject performs 3-5 repetitions short of the prescribed amount, 5 lbs. was removed for the subsequent set. An RIR of 6 or more would call for an addition of 10 lbs. during the subsequent set. If a subject falls 6 or more repetitions short of the prescribed amount,10 lbs. was removed for the subsequent set. This load progression scheme was continued onto the same session for the following week. For instance, during the bench press, if the subject reports a RIR of 3 on the last set during session 1 of week 1, 5 lbs. was added for the first set of the bench press during session 1 of week 2. This strategy was used to ensure proper progression for each subject and to control for individual differences in the rate of strength development. 21

30 Table 1. Resistance Training Protocol EXERCISES MONDAY WEDNESDAY FRIDAY Back squat Bench press 4 sets x 12 reps 4 sets x 10 reps 5 sets x 8 reps Deadlift 4 sets x 12 reps -- 5 sets x 8 reps Shoulder press Seated cable row Dumbbell chest press 3 sets x 10 reps 3 sets x 8 reps 3 sets x 6 reps Dietary Supplementation Protocol The experimental dietary supplement was a multi-ingredient compound comprised of a blend of branch chain amino acids (BCAA) (6g/serving, 4:1:1 leucine, isoleucine, and valine ratio), L-glutamine (3g/serving), beta-alanine (2g/serving), creatine HCl (2g/serving), and black pepper fruit extract (5mg/serving). A single serving provided 1g of carbohydrate constituting 5 kcals. The placebo was a calorie- and taste-matched supplement without the active ingredients listed above. Supplements were in a powder form and mixed in approximately 10-12oz. of water prior to consumption. An outside member naïve to the nature of the study administered the supplements in solid-colored containers labeled X or Z. Information indicating the content of container X or Z was sealed in an envelope and opened once data collection was completed. Subjects then consumed the supplement assigned to their group (EXP consumes MIES, PLA consumes placebo). One serving was consumed immediately following a training session and one serving immediately prior to sleep on training days. During each recovery/rest day (Tuesday, Thursday, Saturday, and Sunday), subjects received 2 single serving packets of their assigned supplement; one serving was ingested with breakfast in the morning, and another serving prior to sleep. 22

31 Laboratory Testing Procedures Maximal Muscular Strength. An assessment of one-repetition maximum (1RM) was administered to evaluate isotonic muscular strength and was adapted and slightly modified from a previous protocol. A 1RM will be defined herein as the greatest load that can be moved through the entire range of motion properly for no more than 1 complete repetition for a given exercise. Investigators administered the 1RM test on the back squat, bench press, and deadlift exercises in the listed order. Participants began testing with ten minutes of self-paced treadmill walking. This was then followed by an exercise-specific warm-up set of 5-10 repetitions at 40-60% of estimated 1RM. After resting 1 minute, participants completed 3-5 repetitions at 60-80% of estimated 1RM. Participants then rest 1 minute and then attempt 90% of estimated 1RM for their first trial. If successful, investigators increased the load based on the participant s perception of the previous attempt; if unsuccessful, the last successful load will define the participant s 1RM. Investigators determined 1RM in 3-5 trials between which participants were provided 3-5 minutes of rest. A rest period of 5 minutes was provided between exercises. The 1RM measurements served to provide reference for baseline measures. Body Composition. Body composition was measured by; dual-energy x-ray absorptiometry (DXA) (Hologic Discovery-QDR Series Densitometer, Bedford, MA) circumference measurements of the upper and lower limb (bicep, waist, hip and thigh), and anthropometric measurements (height, weight, BMI). DXA is a safe and reliable assessment that has been previously implemented in special subject populations (Baim et al., 2005). DXA scans are typically 7-10 minutes in duration and non-invasive with marginal radiation exposure. The effective dose for a whole-body scan using the Hologic 23

32 Discovery system is <5 usv, which is less than half a day of natural background exposure and less than half the dose of a standard x-ray scan. Total body mass was also quantified through the scan. A 3-compartment model of body composition was applied through which FM (kg) and non-bone LBM (i.e. fat free mass bone mineral content) (kg) was analyzed for the whole body. Data for body fat percentage (%BF) was also acquired from DXA measurements. The DXA machine was calibrated before each scan using a manufacturer-provided phantom. All DXA measurements and analyses was conducted by a single certified technologist. Statistical Analyses A 2 (group) x 2 (time) repeated measures analysis of Variance (ANOVA) was used to detect main effects and/or interaction. In the event of a significant F-ratio, a Tukey s post hoc test was used for pairwise comparisons. All statistical analyses were performed using Statistical Package for Social Science (SPSS) with an alpha level set at p<

33 CHAPTER FOUR Descriptive Measures RESULTS Both groups were homogenous based on baseline descriptive measure analysis. There were no significant between group differences for all descriptive baseline measures which included, age, TBM, LM, FM, BF%, height, upper body strength, and lower body strength (Table 2). Table 2. Between-group comparison of descriptive baseline measures Descriptive Measures EXP (n=16) PLA (n=23) p-value Age (y) 21.9 ± ± Total Body Mass (kg) 63.1 ± ± Lean Mass (kg) 44.6 ± ± Fat Mass (kg) 16.1 ± ± Body Fat Percentage (%) 25.6 ± ± Height (cm) ± ± Upper Body Strength (kg kg TBM -1 ) 1.0 ± ± Lower Body Strength (kg kg TBM -1 ) 1.4 ± ± Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. TBM= total body mass Body Composition Measures Total Body Mass There was a main effect for time for TBM (p<0.001) in which post-hoc tests indicated a significant increase from pre- to post-training for EXP (+1.7 ± 0.3 kg, p<0.001) and PLA (+1.5 ± 0.2 kg, p<0.001) (Figure 2). There were no significant group x time interaction for TBM. 25

34 Total Body Mass (kg) * * Figure 2. Pre- to post-training change in Total Body Mass for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. * Significant increase from pre- to post-training (p<0.001) Lean Mass EXP There was a significant main time effect (p<0.001) and group x time interaction for LM (p=0.03) (Figure 3). Specifically, EXP exhibited an increase of 2.1 ± 0.3 kg from pre- to post-training (p<0.001) while PLA demonstrated a 1.1 ± 0.3 kg increase (p=0.001). EXP exhibited a significantly greater change in LM than PLA (p= 0.03). PLA 26

35 ^ * Lean Mass (kg) * Figure 3. Pre- to post-training change in Lean Mass for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. * significant increase from pre- to post-training (p<0.002) ^ significantly greater than PLA (p=0.03) Fat Mass EXP There was a significant group x time interaction for FM (p=0.02) (Figure 4). The pre- to post-training changes in FM was significantly different between groups (EXP= -0.4 ± 0.2 kg vs. PLA= +0.4 ± 0.2 kg) (p=0.02). PLA 27

36 0.8 EXP PLA Fat Mass (kg) Figure 4. Pre- to post-training change in Fat Mass for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. ^ significantly lower than PLA (p=0.02) Body Fat Percentage ^ There was a significant main effect (p=0.02) and group x time interaction for BF% (p=0.01). EXP demonstrated a decrease of 1.2 ± 0.3 % units (p=0.01) while PLA showed no change from pre- to post-training (p=0.41). BF% was significantly lower than PLA post-training (p=0.01). 28

37 1.0 EXP PLA 0.5 Body Fat % (% units) ^ * -2.0 Figure 5. Pre- to post-training change in body fat percentage for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. * significant decrease from pre- to post-training (p=0.01) ^ significantly lower than PLA (p=0.01) Dietary Intake Two way repeated measures ANOVA (2 groups x 6 weeks) revealed no significant main effect or group x time interactions for average daily caloric, protein, carbohydrate (CHO), or fat intake (Figures 6-9). Total average daily intake did not differ between groups for each measure. 29

38 Avg. Daily Caloric Intake (kcal) EXP PLA Avg. Week Figure 6. Average daily caloric intake for each experimental week and total average caloric intake for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group EXP PLA Avg. Daily Protein Intake (g) Avg. Week Figure 7. Average daily protein intake for each experimental week and total average protein intake for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. 30

39 Avg. Daily CHO Intake (g) EXP PLA Avg. Week Figure 8. Average daily carbohydrate intake for each experimental week and total average carbohydrate intake for EXP and PLA Data presented as mean ± SE. CHO= carbohydrate. EXP= Experimental MIES Group. PLA= Placebo Control Group. Figure 9. Average daily fat intake for each experimental week and total average fat intake for EXP and PLA Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. 31

40 Training Volume There was no significant interaction of group x time on total weekly training volume as well as average training volume. Figure 10. Weekly and average total volume between EXP and PLA. Data presented as mean ± SE. EXP= Experimental MIES Group. PLA= Placebo Control Group. 32