Effects of a Multi-ingredient Supplement Study: a Randomized Controlled Trial in Men and Women (SRCT)

Effects of a Whey Protein-based Multi-ingredient Supplement on Muscle Strength, Hypertrophy, and the Acute Satellite Cell Response, in Men and Women: a Double-blinded Randomized Controlled Trial

Resistance exercise training is a known stimulant for muscle protein synthesis (MPS) to occur, however the consumption of protein is also necessary to induce a shift towards a positive net protein balance. Other nutrients have been shown to be useful in the accretion of lean body mass and strength. Whey protein has been shown to improve muscle net protein balance after resistance exercise, more so when in conjunction with creatine. Furthermore, essential amino acids such as Leucine have been found to be important in moderating the shift towards a positive net protein balance, especially post-exercise and in combination with whey protein. Other vitamins and minerals have been shown to aid in permitting muscle protein synthesis rather than stimulating it, but are still necessary for optimal gains in hypertrophy. The effect of a multi-ingredient compound has rarely been investigated, and thus this study aims to combine whey protein, leucine, creatine, calcium citrate and vitamin D and test it in a resistance exercise training program over 12 weeks. Another novel aspect in this study will be testing this supplement against a placebo of equal amounts of protein and amino acids, the placebo will contain collagen protein and the non-essential amino acids alanine and glycine. Finally, this study will recruit both men and women (ages 18-30) to determine possible effects of the supplement on muscle hypertrophy in both sexes. We hypothesize that the synergistic effect of the ingredients in our supplement will induce hypertrophy to a larger degree than resistance exercise training with the placebo, and that these benefits will persist in both male and female participants.

Supplementation in the form of whey protein, creatine and amino acids have all been shown to be a potent stimulus for muscle hypertrophy, when combined with resistance exercise. Burke et al. (2001) reported that males who supplemented with a combination of whey protein and creatine had greater increases in lean tissue mass and strength than those who supplemented with only whey protein or placebo. Hulmi at al. (2010) identified the most important component of whey to be its high concentration of the branched chain amino acid leucine, a critical amino acid for increasing muscle protein synthesis and suppressing muscle protein breakdown. The combination of leucine and whey protein has been shown to promote significant increases in muscle cross-sectional area. Vitamin D and calcium citrate have also been shown to enhance increases in muscle strength and lean mass, albeit in a permissive rather than stimulatory manner. Protein supplements have consistently been shown to promote greater gains in hypertrophy than their placebo counterparts, however these placebo supplements are often isoenergetic carbohydrate-based formulas. Few studies have compared two supplements with an equal amount of protein and amino acids, or have combined multiple beneficial ingredients to form a multicomponent supplement to promote greater gains in lean body mass and strength. Finally, sex differences in resistance training programs have rarely been studied, less so when testing a new supplement. Thus, the purpose of this study will be to determine whether there is an augmented effect of our nutritional supplement on the degree of hypertrophy in young male and female adults involved in a resistance training program. This nutritional supplement will be compared with a placebo containing an equivalent amount of collagen protein and non-essential amino acids. A secondary analysis will assess sex-based differences in strength and muscle size. Based on previous literature, we hypothesize that the combinatorial effect of whey protein, leucine, creatine, calcium citrate and vitamin D will induce hypertrophy to a larger degree than resistance exercise with the placebo in young adults. A synergistic combination of ingredients with known benefits will be given twice daily to healthy young men and women (18-30 years old), (n = 22) in conjunction with a 12-week undulating periodized resistance training protocol, in which the repetition number and intensity of the training sessions will vary. Notable ingredients in this supplement include whey protein isolate (20g), leucine (2g), creatine monohydrate (2.5g), calcium citrate (300mg) and vitamin D (1000IU). The placebo condition, also comprised of males and females (18-30 years old), (n=22) will ingest a formulation containing an equivalent amount of collagen protein (20g), as well as an equivalent amount of amino acids (alanine, 1.4g and glycine, 0.6g). Pre- and post- assessments will include dual-energy x-ray absorptiometry (DEXA), one-rep maximum (1RM), and an ultrasound of the quadriceps. Four muscle biopsies will be taken from the vastus lateralis: 1) at rest prior to training, 2) 48h after an acute bout of exercise prior to training, 3) at rest post-training, and 4) 48h after an acute bout post-training. This allows for comparisons of acute and trained effects of both conditions. Using novel immunohistochemical staining procedures we will examine the resistance exercise-induced changes in fibre type distribution and fibre cross-sectional area in the supplement and placebo conditions as well as perform sex-based comparisons. The novel aspect of this study will be the comparison of two isoenergetic, isonitrogenous supplements as stimulants for muscle protein accretion, as opposed to using carbohydrates as a placebo. The supplement will contain a new formulation of ingredients yet to be studied together. The synergistic effect of these ingredients will attempt to maximize muscle growth in both males and females, meaning that associations between sex and exercise can be assessed. This research will help us identify a new method to augment resistance exercise training through protein, amino acids, vitamins and minerals as well as expand the literature surrounding the effects of these ingredients on muscle hypertrophy.

Each subject's supplement is coded with a different number, and neither the researchers nor the subjects are aware of the condition the subject is placed in.

Participants in the experimental condition will consume a supplement containing Whey Protein Isolate (20g) and other ingredients

Participants in the experimental condition will consume a supplement containing Collagen protein (20g) and other ingredients

Histochemical methods will be used to determine how the supplement and training affect muscle growth and regenerative capacity, specifically the behaviour of satellite cells.

1RM testing pre- and post-training will be compared to determine how the supplement and training affect muscular strength

Histochemical methods will be used to determine how the supplement and training affect fiber type distribution (% type I vs. % type II fibers pre- and post-training)

Inclusion Criteria: Aged 18-30 years old Healthy based on questionnaire responses (see exclusion criteria) Recreationally active (exercising ~2x/week) with some resistance training experience (no more than 2 times weekly) allowed Exclusion Criteria: Any acute or chronic illness, cardiac, pulmonary, liver, or kidney abnormalities, uncontrolled hypertension, insulin- or non-insulin dependent diabetes or other metabolic disorders-all ascertained through medical history screening questionnaires Arthritic conditions Individuals who consume any analgesic or anti-inflammatory drug(s), prescription or non-prescription, chronically will be excluded A history of neuromuscular complications Individuals on any medications known to affect protein metabolism (i.e. corticosteroids, non-steroidal anti-inflammatories, or prescription strength acne medications). Extensive history of resistance training in the year prior to study entry. Answers 'yes' to any question on the screening questionnaire

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Wageh M, Fortino SA, McGlory C, Kumbhare D, Phillips SM, Parise G. The Effect of a Multi-ingredient Supplement on Resistance Training-induced Adaptations. Med Sci Sports Exerc. 2021 Aug 1;53(8):1699-1707. doi: 10.1249/MSS.0000000000002641.