22g High EAA (6g) Protein + 36g CHO Pre- / Intra-Workout Boost Fat Oxidation & PWO Resting(!) Energy Expenditure

I don't doubt that you can do that, too!

It does sound awkward: If you mix Twinlab: Amino Fuel (22 g protein - 6 g essential amino acids | L-phenylalanine: 633 mg; Lvaline: 781 mg; L-tryptophan: 133 mg; L-threonine: 679 mg; L-isoleucine: 565 mg; L-methionine: 292 mg, L-histidine: 282 mg; L-leucine: 1350 mg; L-lysine: 1449 mg) with a regular  sports recovery drink that contains 36g of simple sugar, down half of the resulting 800ml serving of whatever you want to call this mix immediately before your workout and consume the rest during the rest periods between sets, this will have measurable effects on your resting energy expenditure and fat oxidation.

From long-term to short time effects

At first, it does questionably sound counter-intuitive that the ingestion of an EAA + carbohydrate mixture before / during would increase the resting energy expenditure and rate of fatty acid oxidation after your workout. On the other hand, if you think about the long-term effects of corresponding supplement regimen, you don't have to look far, to find evidence that they can promote both, muscle gain and fat loss (Bird. 2006).

You can learn more about protein intake at the SuppVersity

Are You Protein Wheysting?

Cod protein for recovery

Protein requ. of athletes

High EAA protein for fat loss

Fast vs. slow protein

Too much ado about protein?

Kyle J. Hackney, Andrew R. Kelleher, and Lori L. Ploutz-Snyder from the Syracuse University speculated that the highly beneficial changes in body composition Bird et al. observed in their study participants over the course of a 12-week strength training + EAA & CHO supplementation that after "[t]hese adaptations may be related to the acute energy expenditure and substrate utilization responses in the postexercise period." (Hackney. 2013)

Figure 1: The changes in body composition (in kg) in response to 12 weeks of resistance training + placebo, CHO, EAA or CHO + EAA supplementation in 2006 study by Bird et al. "inspired" Hackney et al.

Against that background, it was only logical to conduct a study to examine how multiple bouts of resistance exercise with and without the strategically timed intake of amino acids affect the resting energy expenditure (REE) and respiratory exchange ratio (RER). The results could after all explain if the long/er) term effects on body composition that have been observed in previous studies using chronic training and supplementation regimen are maybe nothing but necessary consequences of repeated acute increases in REE or decreases in RER (you hopefully remember that a decrease in the respiratory exchange ratio signifies an increase in fatty acid oxidation).

Experimental design and results

To this ends, the researchers recruited 10 young (mean age: 23.4y) recreationally trained male participants. All of them had been participating in general resistance training exercise for a minimum of 3 days per week for at least 6 months.

Figure 2: Changes in resting energy expenditure (kcal/day) and comparison of training volume in 58g CHO (black bars) and EAA + CHO (white bars) trials (Hackney. 2013).

As you can see in Figure 2, Hackney et al.'s original hypothesis that "intake of amino acids with each resistance exercise session would lead to greater perturbations of REE and RER" (Hackney. 2013) does unquestionably hold for this population of average (rookie) gymrats.

Whether the scientists "main finding" (Hackney. 2013), i.e. the 3.61% increase in resting energy expenditure (REE) will be similarly pronounced in advanced trainees is yet as questionable as the real-world effects of this artificial value. Despite the fact that Hackney et al. are right, when they say that our resting energy expenditure "represents the largest component of [our] total daily energy expenditure (60–85%) and has been implicated as a major contributor to overall body mass management " (Hackney. 2013), I am not sure how "major" an increase of only 66kcal per day actually is... I mean,  if this pathetic increase in resting energy expenditure was the actual driving force we would need almost 100 days to shed a hilarious pound of body fat (note: the reason I use the flawed 3,500kcal = 1lbs of fat rule of thumb here is that the whole REE calculations would be pointless if you didn't put at least some faith into the "energy in vs. energy out" hypothesis of weight loss - right?)

SuppVersity Suggested Read: "Fat Loss Principles That Work: 10g+ of EAAs W/ Every Meal. Do Energetic Costs of Protein Synthesis Trigger This Effect?" |  read more

Bottom line: It stands out of question that your training success can benefit from a high EAA protein source and some carbs you consume before and during the exercise session. Whether the fat loss benefits are actually brought about by the marginally increased resting energy expenditure (REE) is yet something I doubt - it certainly helps fat loss, but clearly isn't its main motor.

Don't get me wrong, this does not imply that you will benefit from this type of "peri-workout" supplementation. And let's be honest, the end most of you probably don't care about the exact underlying mechanisms, as long as your body composition keeps improving, right?

References:

• Bird, S. P., Tarpenning, K. M., & Marino, F. E. (2006). Independent and combined effects of liquid carbohydrate/essential amino acid ingestion on hormonal and muscular adaptations following resistance training in untrained men. European journal of applied physiology, 97(2), 225-238.
• Hackney, K. J., Kelleher, A. R., & Ploutz-Snyder, L. L. (2013). Amino Acid-Carbohydrate Intake Combined with Multiple Bouts of Resistance Exercise Increases Resting Energy Expenditure. ISRN Nutrition, 2013.

Leucine, Citrulline or a Non-Essential Amino Acid Mix - Which Amino Acid(s) are Most Effective in Preventing Muscle Loss During an 18h (Intermittent) Fast?

Image 1: If Chris, "the Techician", Aceto's usually well-informed sources are right and the former Mr Olympia Jay Cutler is currently trying to lose muscle (I heard him say that on Heavy Muscle Radio), Cutler would be ill advised if he ingested ~20g of non-essential amino acids during and / or in-between extended fasts and hours of arduous low-intensity cardio sessions (img  MuscleTech)

Those of you who followed the "Amino Acids for Super Humans" series I did earlier this year on Carl Lanore's Super Human Radio may remember the arginine < > citrulline < > ornitine cycle and how I tried to explain that, from a physiological perspective, arginine's role in ammonia detox is probably as, if not more important than its role in the production of nitric oxide. What most of you will probably have overheard, or, in the respective shownotes, over-read, was my reference to a 2006 study from the University of Paris, which was - at least to my knowledge - the first study to show that citrulline (much like leucine) increases protein synthesis and thusly reduces the loss of muscle protein in old malnourished rats (Osowska. 2006). As it is often the case with isolated study results like that, these observations have not gotten much attention within the research community, so that it is not very surprising that the latest information on citrulline's putative role in whole body protein homeostasis come from the same laboratory at the Sorbonne, as the previously cited ones.

Citrulline vs. Leucine, and non-essential aminos as a control!?

What is particularly interesting about these results, the scientists from the Département Biologie Expérimentale, Métabolique et Clinique at the Pharmaceutical Faculty of the venerable Université Paris Descartes published in the (btw. highly recommendable) Journal Amino Acids, is that they allow for a direct comparison of the magnitude and the mechanism the ingestion of citrulline, leucine or a mix of other non-essential amino acids has on the fractional protein synthesis in skeletal muscle tissue (Tibialis anterior) in a fasted state (18h food deprivation).

Figure 1: Fractional protein synthesis (in %/h) in tibialis anterior muscle of fasted rats 50 minutes after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

To my own surprise the winner of the battle of the "protein anabolic amino acids" is neither the usual (leucine), nor the unusual suspect (citrulline), but rather the non-essential amino acid combo which consisted of 1.35g/kg of alanine, glycine, proline, histidine, asparagine and serine.

Alanine, glycine, proline, histidine, asparagine, serine - Non-essential high potentials?

Let's briefly put this surprising result into (a human) perspective: If we assume that you are on an extended intermittent fast, traveling or had - for whatever other reason - no access to food for 18h, then the ingestion of 0.22g/kg of a non-essential amino acid mixture (if you weigh 80kg that would be 17.5g), would induce a 9.37% greater increase in muscle protein synthesis than the same amount of leucine and a 16.67% greater increase than 23g of l-citrulline.

Figure 1: Phosphorylation of Akt, s6K, 4EBP1 (left) and AMPK (right) 60min after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

If we combine the previous calculations with the data from the Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component, it becomes even more obvious that this study provides further evidence against the current over-emphasis of l-leucine which is so prevalaent especially among the bodybuilding-oriented physical culturists. As I have pointed out in previous posts, here at the SuppVersity, pushing the "protein-anabolic gas-pedal" through the floor (=ingesting huge amounts of leucine on its own) makes no sense if your car has long run out of fuel (=there are no amino acids to synthesize).

Against that background it is actually not very surprising that the protein synthesis in the fasted leucine group was reduced, although the phosphorylation of  p70S6K was identical and the one of 4EBP1 even greater (both indicate that the protein synthetic machinery was set into gear) than in the fed control. What is surprising, though, is the fact that the actual protein synthetic response in the leucine group fell 10% short of the one that was observed in the tibialis muscle of the rodents which receive an isonutrogenous amount of non-essential amino acids. After all, previous studies have suggested that the induction of measurable increases in protein synthesis was an exclusive property only branched chain (BCAA) or essential (EAA) amino acid mixtures would posses. Methodological differences in the design of respective studies aside, Servane Lé Plenier and his colleagues suggest the following two possible explanations for the surprising effects the alanine, glycine, proline, histidin, asparagine and serine combo exhibited on skeletal muscle protein synthesis in the fasted state:

[firstly,] in the fasted state, NEAA homeostasis is maintained by catabolism of essential amino acids (EAA) - alanine, for example, is produced in muscle from LEU and pyruvate - and limited EAA availability affects MPS since it is well known that a deficiency in one amino acids may be a limiting step for protein synthesis. Hence, in the fasted state, NEAA administration could spare EAA utilization and thereby preserve MPS.

[secondly,] one or more amino acids in the NEAA mixture could display specific anabolic properties. For example, alanine has been shown to stimulate liver protein synthesis in starved rats (Perez-Sala. 1987), but to the best of our knowledge this effect has not been shown in muscle. Similarly, proline and glycine may possess pharmacological properties that could indirectly modulate protein synthesis.

Personally, I don't believe that any of the non-essential amino acids (NE-AA) in the NE-AA formula actually had an individual effect on protein synthesis beyond its ability to spare essential amino acids and its availability as a substrate for inter-organ amino acid transfer (especially for alanine and asparagine, which are transaminated in the liver, this could be an important factor). So that the practical implications of this study should be clear: if you want to minimize muscle loss during a(n) (intermittent) fast, you better have some non-essential amino acids with your leucine!

One question answered, 999 new ones raised

Image 2: If you have read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will probably already have noticed that the ingestion of non-essential amino acids had the least impact on the fasting-induced increase in AMPK-phosphorylation of all three treatments. And I guess I don't have to tell you that this is good news for all intermittent fasters out there - spare the muscle, improve your health and burn the fat, what more can you as for?

Unfortunately, this study leaves us with way more questions than answers. I personally, for example would venture the guess that the ingestion of a complete EAA product would result in an even more profound amelioration of the fasting induced reduction in fractional protein synthesis. That being said, the latter could also compromise another advantage of the non-essential amino acids, I have not even mentioned, yet: their almost non-existent effect on intra-muscular AMPK-expression (cf. figure 2, right). If you read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will be familiar with notion that the fasting-induced phosphorylation of intra-muscular AMPK is responsible for the majority of the health, as well as the closely related fat-burning effects of (intermittent) fasting. Now, if the ingestion of a ~20g bolus of alanine, glycine, proline, histidine, asparagine and serine could increase your skeletal muscle protein synthesis back to almost normal levels (NE-AA -12.5% vs. leucine-only -20%), while keeping the AMPK-alpha levels maxed out (cf. figure 2, right), it would at least warrant an experiment before we totally discard the possibility that, under certain circumstances, such as the fasting window of an intermittent fast, the oftentimes disregarded "non-essential amino acids" could perhaps be more than just a band-aid when you have run out of essential ones.

Whether there will be a place for citrulline in particular is questionable, though. With the least effect on protein synthesis and the greatest impact on AMPK, it would de facto be a "band-aid" solution, for everyone who fasts, deliberately. In other contexts, however, l-citrulline supplementation could well have its merits. In cancer patients it could for example be used to ameliorate muscle loss without triggering the pro-carcinogenic (Garcia-Maceira. 2009), but I guess this would be the topic of another study and another blogpost, here at the SuppVersity ;-)

Fat Loss Principles That Work: 10g+ of EAAs W/ Every Meal. Do Energetic Costs of Protein Synthesis Trigger This Effect?

EAAs beyond whey: It may not necessarily look like this, but this salad (repicecorner) is an EAA power horse with cheddar cheese (25% protein, 0.49 EAA / P ratio), tuna (in oil, 29%,  0.45) and kidney beans (9%, 0.45). You see, it does not always have to be chicken breasts or whey to get beyond the 10g+ EAA threshold, I have repeatedly suggested as one of the fundamental rules of dieting for weight loss, maintenance and muscle gain.

Many people take it for granted that you become fat, when you get old. If you look at the statistics, you could even make a point that obesity has some protective effects with esp. with respect to CVD mortality. Scientists call this the "obesity paradox" (Kastorini. 2012). What's particularly paradox, at least in my humble opinion, is yet not the phenomenon itself, but rather the fact that it gets smart scientists derailed from working on useful dietary and exercise interventions to prevent the development of heart disease, cancer, metabolic syndrome etc. in early years. Instead, they argue ex-post, i.e. when the baby has already been thrown out with the bathtub by comparing sick lean (in parts even cachectic) and sick "obese" people, why their statistical shenanigan that's based on the useless BMI produces paradoxical results. And that, when studies such as the one Jacobs et al. did in 2010 clearly show that 50+ year old men and women with waist circumference >120cm and >110cm, respectively, have 2x higher all-cause mortality risk than their lean peers - irrespective of BMI (Jacobs. 2010)!

To get lean and stay lean, yet not thin and skinny fat is therefore a challenge everyone...

... from the child in the Kindergarten to the obese granny in the nursing home is facing. Against that background a previous study by Loenneke et al. comes to mind. The results of their analysis, which were published in Nutrition and Metabolism in January 2012 clearly show that the amount of times people eat meals with a 10g+ EAA content per day was inversely related to percent central abdominal fat (Loenneke. 2012). In previous studies EAAs have also been shown to improve glucose clearance without increases in insulin and in the absence of effects on the fat burnin and health promoting expresion of AMPK-alpha2 in skeletal muscle tissue (see "EAAs Stimulate Muscle Glucose Uptake by Exponentiating Insulin's Effect on GLUT4 Expression"). With the advanced publication of a study by Coker, Miller, Schutzler, Deutz and Wolfe in the online verison of the Nutrition Journal a couple of days ago, the notion that EAAs have a particularly beneficial effect on fat loss - in this case in obese elderly individuals - gets further support from a well-controlled randomized trial (Cooker. 2012).

EAA-rich protein increases fat loss to a greater extent than low EAA protein

The researchers from the Center for Translational Research in Aging and Longevity and the University of Arkansas for Medical Sciences in Little Rock, AR, USA randomized 12 elderly individuals (mean age 69 years) to an 8 week, caloric restriction diet utilizing equivalent caloric meal replacements (~850 kcal/day; the exact nutrient composition can be found in figure 1) + ~400kcal from solid foods (total intake: ~1,250kcal/day; the subjects were free to chose their solid meals but were provided with a list of examples the should pick from, if possible).

Figure 1: Macronutrient composition of the meal replacements used in the study (Cooker. 2012)

The diet was designed to induce a 7% weight loss in two months. And while both,  the rate of weight loss (~1.6lbs per week), as well as the relatively high caloric deficit are certainly appropriate for someone with a 30+ BMI and ~40% body fat, leaner people will fare better with a less pronounced kcal deficit or (alternatively) have to add some strategically planned refeeds to the equation in order to minimize the loss of lean mass and, more importantly, avoid the ensuing reduction in energy expenditure (for the obese, the latter is actually less of the problem, because the downsides of being calorically deprived are at least partly counglucose tolerance and leptin sensitivity with every gram of body can actually help the body recognize that there is still plenty of energy that has just not been available (glucose) or "visible" (fat) before).

Figure 2: Changes in lean and fat mass (kg, left) and fractional protein synthesis rates (FSR) in participants receiving iso-caloric meal replacements with identical macronutrient compositions (see figure 1), but different amounts of essential amino acids (EAAs) content (Coker. 2012)

As the data in figure 2 goes to show you even the obese individuals in the study at hand lost a non-negligible amount of lean mass - unfortunately the body composition was measure with a sophisticated, but still body impedance based device, the trends are still accurate, but it is questionable in how much we are actually talking about ~2 and 2.5kg of muscle mass (figure 2, left), because somebody's "lean body mass" does obviously include more than just skeletal muscle.

When it comes to supplements, we are often like children on Christmas eve. About all the new stuff we get we tend to forget our former favorite and often way more fun to play with toys. Don't make this mistake and ditch your PWO whey (personally, I like a ~1.5:1 whey + micellar casein mixture) for EAAs, they don't come close... read more

Be that as it may - since the before and after values were taken with the same device the changes should be correct, so that both the slightly yet not statistically significantly ameliorated loss of lean body mass and, more importantly, the significantly higher degree of body fat loss in the EAA meal replacement (EAAMR) group speak in favor of the 5 servings of a the 170 kcal, 6g EAA per day. Moreover, "the sparing influence of muscle loss might have been demonstrated with a larger sample size", so that you can take it for granted that the preservation of precious muscle mass is an advantage of being choosy with your protein sources and preferring those with higher over those with lower essential amino acid contents.

On a related note: I don't know if you noticed, but with a total energy content of 850kcal and 30g EAA these 5 meal replacements did in fact have exactly those 10g+ of essential amino acids, I have repeatedly recommended to have with each of the 3 meals most people consume in the course of the day.

In all fairness, it should also be mentioned that despite not being significantly different at baseline, the body fat percentage of the subjects in the EAA meal replacement group was ~3% higher to begin with.This may seem irrelevant, since figure 2 compares lean mass and fat mass as absolute changes and not their percentages, but in the end, the amount of fat you you can drop within a given time-frame decreases with lower body fat percentages.

Do the energetic costs of protein synthesis drive fat loss?

Another interesting observation Coker et al. made is the close association between fat loss, on the one hand, and increased protein synthesis (55%), on the other hand. The researchers take this as an incentive to do one of the of the much loved calories in vs. calories out calculation and come up with the following hypothesis:

"Acute administration of EAAMR did promote a significant increase in skeletal muscle protein FSR compared to CMR. Assuming that the energy cost of protein synthesis is 3.6 kJ/g and the baseline GAIA-derived lean tissue mass was 56.4 kg for EAAMR and 54.4 kg for the CMR, we can extrapolate that the overall energy discrepancy between the two groups was roughly equivalent to 27,170 kcal or 3.5 kg of weight loss across the entire caloric restriction-based weight loss paradigm. Based on the amount of total lean mass in each group, this value takes into account a consistent intervention structure of five servings/day across an eight week period. In short, these calculations suggest that differences in the source of intact protein/formulation of EAA may have a significant influence on diet-induced energy expenditure that coincides closely with the greater reduction of adipose tissue in EAAMR compared to CMR." (my emphasis in Coker. 2012)

I usually discard fallacious calculations like this one if they are not highlight the stupidity of trying to eat exactly as much as some funky formula + the figure on your treadmill, pedometer, heart rate monitor or whatever fancy tool you may use to "measure" your energy expenditure suggest you would have burned in the last 24h. In this case, however, I made an exception, because I feel that the notion that protein quality is one of the myriad of parameters that are missing from this foolish calculation is important, for lean and obese people from all age groups who are trying to shed body fat.

Bottom line: The take away message of the study is in the end identical to the previously mentioned study by Loenneke et al.: Make sure you hit the 10g EAA threshold with each and every of your meals, if being lean and muscular not skinny yet fat is your goal.

References:

• Coker RH, Miller S, Schutlzer S, Deutz N, Wolfe RR. Whey protein and essential amino acids promote the reduction of adipose tissue and increased muscle protein synthesis during caloric restriction-induced weight loss in elderly, obese individuals. Nutr J. 2012 Dec 11;11(1):105. [Epub ahead of print]
• Jacobs EJ, Newton CC, Wang Y, Patel AV, McCullough ML, Campbell PT, Thun MJ, Gapstur SM. Waist circumference and all-cause mortality in a large US cohort. Arch Intern Med. 2010 Aug 9;170(15):1293-301.
• Kastorini CM, Panagiotakos DB. The obesity paradox: methodological considerations based on epidemiological and clinical evidence--new insights. Maturitas. 2012 Jul;72(3):220-4.
• Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2012 Jan 27;9(1):5.

No Advantage of Bolus Ingestion of EAAs in Young Men!? Cereal Bread Not Better for Weight Control. Saturated Fat & the Heart. Plus: Serine for Your Weekend Alcohol Binge!

The "muscle full effect" indicates you don't have to consume 4 scoops at once.

With the publication of the latest issue of The Journal of Nutrition came a handful of interesting scientific papers I will briefly introduce in today's SuppVersity Nutrition Science Update.

The corresponding studies deal with the link of saturated fat to heart disease (Puaschitz. 2014), the effects of proteinogenic amino acid serine (one of the non-essential amino acids) on homocysteine metabolism in a rodent model of alcoholic fatty liver disease (Sim. 2014).

And when we're through with those, we will take a closer look at the effects of cereal enriched breads on the appetite ratings and postprandial glucose, insulin, and gastrointestinal hormone responses related to hunger and satiety in healthy men and women (Gonzalez-Anton. 2014), and the "muscle full effect", or rather limits to maximal protein synthesis in man (Mitchell. 2014).

Read more short news here at the SuppVersity

Obesity Research Upd. Nov. '14

Exercise Res. Upd. Nov '12(1)

Exercise Res. Upd. Nov '12(2)

Nutrition Res. Update Nov. '14

Weight Loss Tricks & More

Reductive Stress, Iron & the Military

• Saturated fat and your heart - Right from the Haukeland University Hospital in Norway comes a new study that investigated the associations between self-reported dietary SFA intake and risk of subsequent coronary events and mortality in patients with coronary artery disease (CAD).
  
  The study included patients who participated in the Western Norway B-Vitamin Intervention Trial and completed a 169-item semiquantitative food-frequency questionnaire after coronary angiography - 2412 patients, total, 81% men, 19% women with a mean age of 61.7 y.
  After a median follow-up of 4.8 y, a total of 292 (12%) patients experienced at least one major coronary event during follow-up.  And while a gigh intake of SFAs was associated with a number of risk factors at baseline, "there were no significant associations between SFA intake and risk of coronary events [age- and sex-adjusted HR (95% CI) was 0.85 (0.61, 1.18) for the upper vs. lower SFA quartile] or any secondary endpoint. Estimates were not appreciably changed after multivariate adjustments" (Puaschitz. 2014).
  

  

  Figure 1: Hazard ratios according to % saturated fat intake of total energy intake compared to minimal saturated fat intake (HR = 100%) in 2412 subjects (Puaschitz. 2014).

  In other words, if you ask researchers from Northern Europe, their answer to the question, whether our high intake of saturated fats is the reason we are dying prematurely from heart disease is "no". This stand in line with a recent review of the current evidence by O'Keffee et al. who point highlight that the different results (which often depend on the country, where the studies are conducted) may be attributable to the fact that "not all SFA are created equal and the food sources of SFA". Accordingly the researchers from the King's College in London, the Luke’s/Roosevelt Hospital, the New York Nutrition Obesity Research Centre and the Columbia University in New York recommend that "individual characteristics of the SFA, such as chain length, should be considered in dietary recommendations" (O’Keeffe. 2014)... and I would like to add: In every future study, as well.
  
  I mean, this and the foods from which the subjects in the study at hand got the majority of their saturated fat intake may well be the reason that there was a statistically significant correlation between high fat intakes and the occurernce of coronary artery disease (remember: all participants had CAD, already) in the cohort Western Norway B-Vitamin Intervention Trial.
• L-Serine as super-supplement for binge drinkers? At least in rodents the provision of 200mg/kg body weight (for humans this would be ~1.2-1.5g/day) serine in the diet led to an attenuation of alcohol-induced increases in serum homocysteine and hepatic triglyceride (TG) concentrations (>5-fold in the control mice) by 60.0% and 47.5%, respectively.
  

  

  Figure 2: Liver triglyceride levels, serum ALT and serum homocysteine levels in control mice (C) and "binge drinking mice" (EV) with and without 20mg/kg (ES20) and 200mg/kg (ES200) serine in their diets (Sim. 2014)

  Moreover, in the chronic ethanol study, l-serine also decreased hepatic neutral lipid accumulation by 63.3% compared with the ethanol group and ramped up the glutathione and S-adenosylmethionine content of the liver by 94.0% and 30.6%, respectively.
  
  If we assume that serine is only half as powerful, when it is given to humans, I would recommend you drink your Vodka Red Bull with serine in the future ;-)
• Super-satiating cereal enriched breads - I guess "super-satuating" is an exaggeration, but there is no doubt that the addition of variety of cereal flours (wheat, oat, and spelt) and 22% dried fruits (figs, apricots, raisins, and prunes) to regular bread lead to a significant improvement of appetite control by reducing hunger and enhancing satiety in 30 healthy adults (17 men and 13 women) aged 19–32 y with body mass index of 19.2–28.5 who participated in an experiment that was conducted at the University of Granada in Spain (Gonzalez-Anton. 2014).
  

  

  Figure 3: The hormonal changes would indicate increased satiety, the subjects reported increased satiety, but their 4h energy intake was identical in both condition (Gonzalez-Anton. 2014)

  Whether the decrease in prospective consumption and increased satiety is enough to have long-term benefits on weight control is yet questionable, because the subsequent ad libitum energy intake in a 4 h period after the ingestion of the "enriched" bread did not differ from that in the control condition, even though the postprandial blood glucose, insulin, ghrelin, were lower and the pancreatic polypeptide AUC (an indicator of satiety) was higher than with the control bread.
  
  Speaking of insulin: In view of the fact that the latter actually is a satiety hormone and its release is closely related to glucagon-like peptide (GLP) 1 and gastric inhibitory polypeptide (GIP) where the AUC (areas under the curve) were lowered as well, it's eventually maybe not too surprising that the "enriched" bread was not better than the regular one.
• Muscle full? What's limiting protein Synthesis? Scientists from the Clinical, Metabolic, and Molecular Physiology, MRC–Arthritis Research UK Centre of Excellence for Musculoskeletal Ageing Research at the University of Nottingham and the Royal Derby Hospital in the United Kingdom recently determined the effect of Bolus (=all the aminos at once) vs. Spread EAA feeding in young men, hypothesizing that muscle-full is regulated by a dose-, not delivery profile–, dependent mechanism; and what they found was surprising for us - not for the researchers, though:

  

  Figure 4: Even though the study was conducted in young men, the overall dosage of 15g may potentially have had an effect on the outcome. On the other hand: If you "overdose" it would actually be more likely for spread protein ingestion to have superior effects. Against that background the "low" dose of "only" 15g of pure EAAs is not an argument that would falsify the results of the study at hand (Mitchell. 2014)

  "Despite distinct plasma and muscle profiles, Bolus feeding provided no anabolic advantage over Spread feeding (or vice versa); these findings are in keeping with our hypothesis of there being an intrinsic muscle-full state in young men at rest.
  
  Bolus feeding led to rapid aminoacidemia with a brisk upstroke and high peak plasma EAA and leucine concentrations. Spread feeding, by comparison, resulted in lower, later peak concentrations. Despite this, identical MPS responses were observed, even with the same latency (of ~90 min) and amplitude.
  
  Furthermore, with both feeding strategies, basal MPS was observed 180 min after consumption of either Bolus or the initial Spread doses. This preceded the peak Spread plasma EAAs, in keeping with the onset of a muscle-full state.

  As the scientists point out, their results do thus "suggest that, in healthy young men, it is dose dependent mechanisms that regulate the size of the anabolic response to feeding and that this response" and that this dose-dependent anabolic response "is not perturbed by later arriving, lower-amplitude aminoacidemia." The researchers also highlight hat it would seem "vital to have such a mechanism in place"; because of the "stability of muscle mass from year to year in healthy younger populations" (Mitchell. 2014). Eventually, the differences may well be explained by the existence of three distinct phases in the postprandial period, the scientists argue:
  

  

  Figure 5: Absolute changes in FSR from fasted (2120 to 0 min) to fed (0 to 240 min) (A), actual FSRs (B) and plasma EAA and insulin concentrations, phospho- 4EBP1 Thr65/70 and muscle protein synthetic rates, normalized to their own data spans shown on the same axis (C and D) in young men after consumption of 15 g of mixed-EAA meals by Bolus or Spread treatment. The black arrows represent ingestion of 15 g EAAs once, and the gray arrows represent ingestion of 3.75 g EAAs 4 times (Mitchell. 2014)

  "After the onset of essential aminoacidemia, a latent period exists when a significant negative arteriovenous EAA balance is detectable (Mitchell. 2013) but incorporation of EAAs into newly synthesized myofibrillar proteins is not. The existence of a similar latent period in response to Bolus and Spread EAA ingestion suggests that providing time for adequate intracellular EAA accumulation, even with rapid aminoacidemia with Bolus, is crucial before MPS can be ‘‘switched on.’’ After this latent period, a transient stimulation in MPS, lasting ;90 min (Bohé. 2001), occurs before the onset of the muscle-full state restores basal MPS despite sustained, near-peak postprandial EAA availability" (Mitchell. 2014).

  Put simply, it takes long enough for the muscle protein synthesis to gain full speed to incorporate all the amino acids the healthy subjects received in 4x45min boluses.
  
  Practically speaking this does not necessarily mean that you should give up your previous protein feeding strategies. With intact proteins, of which you know that they are more than the sum of their EAA parts (see "Whey Beyond Brawn"), studies by Moore et al. (2012 | learn more) and Burke et al. (2012 | learn more) yielded different results... albeit with less frequent biopsies that were taken across the postprandial period and thus a lower temporal resolution that does not exclude that said studies simply overlooked the dose-dependency of the muscle-full effect Mitchell et al. demonstrate in the study at hand.

10+ Things You Probably Didn't Know Whey Protein | more

So what are the take home messages from today's research update? I guess the one you will be most interested in, is the related to the Mitchell study which indicates that protein timing and / or the importance of bolus ingestions may previously have been overrated - at least in the short run. We should not forget, after all, that this is a result that would stand in line with Alan Aragon's & Brad Schoenfeld's recent review (Aragon. 2014  on nutrient timing which found a significant effect for the amount of protein people consume, but no evidence of the purported importance of protein timing.

This is yet not the only myth that is tumbling. The idea of heart disease triggering saturated fats and the notion that you could make bread a superfood by adding cereals and dried fruits did not get away unscathed either. With the impressive effects of serine in the rodent study by Sim et al. (2014), we do have another myth to bother with - one of which I would like to remind you that it has to remain a myth until the results have been confirmed in human beings | Comment on Facebook!

References:

• Aragon, Alan Albert, and Brad Jon Schoenfeld. "Nutrient timing revisited: is there a post-exercise anabolic window." J Int Soc Sports Nutr 10.1 (2013): 5.
• Bohé, Julien, et al. "Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids." The Journal of physiology 532.2 (2001): 575-579.
• Burke LM, Hawley JA, Ross ML, Moore DR, Phillips SM, Slater GR, Stellingwerff T, Tipton KD, Garnham AP, Coffey VG. Preexercise aminoacidemia and muscle protein synthesis after resistance exercise. Med Sci Sports Exerc. 2012 Oct;44(10):1968-77.
• O’Keeffe, Majella, and Marie-Pierre St-Onge. "Saturated Fat and Cardiovascular Disease: A Review of Current Evidence." Current Cardiovascular Risk Reports 7.2 (2013): 154-162. 
• Mitchell, William Kyle, et al. "Development of a new Sonovue™ contrast‐enhanced ultrasound approach reveals temporal and age‐related features of muscle microvascular responses to feeding." Physiological reports 1.5 (2013). 
• Mitchell, William Kyle et al. "A Dose- rather than Delivery Profile–Dependent Mechanism Regulates the ‘‘Muscle-Full’’ Effect in Response to Oral Essential Amino Acid Intake in Young Men."J. Nutr. February 1, 2015
• Moore DR, Areta J, Coffey VG, Stellingwerff T, Phillips SM, Burke LM, Cléroux M, Godin JP, Hawley JA. Daytime pattern of post-exercise protein intake affects whole-body protein turnover in resistance-trained males. Nutr Metab (Lond). 2012 Oct 16;9(1):91.
• Puaschitz et al. "Dietary Intake of Saturated Fat Is Not Associated with Risk of Coronary Events or Mortality in Patients with Established Coronary Artery Disease." J. Nutr. February 1, 2015 jn.114.203505
• Sim, et al. "l-Serine Supplementation Attenuates Alcoholic Fatty Liver by Enhancing Homocysteine Metabolism in Mice and Rats." J. Nutr. February 1, 2015 jn.114.199711.

Branched Chain Amino Magic: Study Takes Another Step Towards a Better Understanding of the Anabolic & Anticatabolic Effects of BCAAs and Their Essential Cousins

Image 1: Without the other essential amino acids (EAAs), the branched chain amino acids, leucine, isoleucine and valine (BCAAs) have nothing to "build" your muscle from ;-)

Usually, I do not get very excited, when I hit upon another study into the "protein-synthetic response" that is triggered by the ingestion of branched chain amino acids (BCAAs). I mean, let's be honest... we all know that their ingestion will trigger the phosphorylation of the mammalian target of rapamycin and thusly increase protein synthesis, so why would we need another study where instead of a 17.5% increase in protein synthesis, we would see a 18.3% increase? Actually, we don't... the data Marcus Borgenvik, William Apró and Eva Blomstrand from the  Åstrand Laboratory, Swedish School of Sport and Health Sciences and the Karolinska Institutet, in Stockholm, Sweden (Borgenvik. 2011), collected goes yet well beyond what we have seen in most of the previous studies and is thus well worth an individual blogpost here at the SuppVersity.

BCCAs work! How? Little do we know...

If we are honest, we must concede that our (=the scientific) understanding of the complex processes that are triggered when "large" amounts of BCAAs hit our bloodstream, is very limited. What we know is that we can measure increases in mTOR-expression that correlate with likewise measurable increases in protein synthesis. What we do not really know is how exactly one leads to the other and where the influences of amino acid supplementation and exercise training overlap. This is even more true for the complementary side of the protein synthetic equation of which Borgenvik et al. state that

[w]hereas extensive evidence for the stimulatory effect of amino acids, either alone or in combination with exercise, on protein synthesis has been reported, their effect on protein breakdown is elusive.

In that, it is particularly confusing that "previous investigations involving ingestion of essential amino acids (EAA) in connection with resistance exercise have revealed no attenuating effect on protein breakdown", while studies which investigated the effect of BCAA or leucine in isolation, report reduction in protein degradation in subjects at rest or performing eccentric endurance exercise (MacLean. 1994). Reason enough for the Swedish scientists to recruit a group of seven healthy, recreationally active participants (5 men, 2 women; 27 (± 2) years; height 175 (± 5) cm; weight 67 (± 7) kg), put them on a standardized diet (17% protein; 25% fat; 57% carbs; ~2100kcal for women, ~2700kcal for men) for two days and, on the subsequent morning (subjects reported to the lab fasted) and after a thorough warm-up, have them perform

• 4 sets of 10 repetitions at 80% of their predetermined 1 RM, followed by another 
• 4 sets of 15 repetitions at 65% of their 1 RM of single-legged leg presses.

The subjects used the same leg on all exercises and rested ~5min after each set. Before the warm-up, during and immediately after and 15 and 45min after the exercise regimen the subjects consumed either

• 150 mL of BCAAs (2:1:1 ratio) in flavored water, or
• 150 mL flavored water alone.

The total amount of BCAAs was 85mg/kg or 5.695g for the "average" study participant. After four weeks the experiment was repeated with each participant receiving the opposite treatment.

Figure 1: Complete analysis of serum amino acid levels in the trained and untrained leg of subjects receiving BCAA or Placebo supplement before, during and after the completion of a standardized single-legged leg press exercise (data adapted from Borgenvik. 2011)

As far as the study protocol goes this is thus certainly not an extraordinary study. If you take a look at figure 1, where I deliberately plotted all the data the scientists gathered as far as serum amino acid concentrations are concerned, you will yet realize that what makes this study stand out is the sheer amount of parameters Borgenik et al. have analyzed. Similar data is also available for the amino acid concentrations in the exercised muscle and though, the scientists, who set out to investigate the effects of BCAA supplementation on protein breakdown, would probably disagree with me, here, I feel that this data actually has the most real world significance for physical culturists, like you and me.

Figure 2: Relative increase / decrease in intra-muscular BCAAs and other EAAs in BCAA supplemented subjects vs. placebo control at different time-points before, during and after single-legged leg presses (data adapted from Borgenvik. 2011)

After all, a brief glance at the effects the ingestion of ~6g of BCAA had on the respective tissue levels of leucine, isoleucine and valine (figure 2, BCAA) and the other, "missing" essental amino acids (figure 2, EAA - BCAA) should suffice to understand that though BCAAs may be the necessary to trigger protein synthesis, they are yet obviously not sufficient to "build muscle" - or how else would you explain the

pronounced reduction in the concentration of the aromatic amino acids, tyrosine and phenylalanine, in both plasma and muscle as well as muscle EAA (BCAA excluded) during the recovery period

Borgenik et al. observed in their study? The scientists at least conclude that

[s]ince tyrosine and phenylalanine are neither synthesized nor degraded in skeletal muscle, reduction in the levels of these amino acid could be indicative of an improved net muscle protein balance, i.e. an enhanced rate of synthesis and/or decreased rate of breakdown [and] could  be explained by incorporation into protein.

The accrual of muscle mass (whatever that may eventually mean, cf. yesterday's installment of the Intermittent Thoughts) thusly obviously relies on the presence of all essential amino acids and not just the "branched chained holy grail" of protein synthesis, of which the current study revealed that they (BCAA ingestion) reduced the expression of MAFbx, which regulates the protein transcription factor MyoD and the eukaryotic initiation factor-3f (eIF-3f), which, in turn is of importance in the mTOR-p70S6k signaling pathway, by 30% and 50% in the resting and exercising legs, respectively.

Figure 3: Relative (compared to placebo) mTOR and p70S6K phosphorylation in response to BCAA supplementation in exercised (EX) and non-exercised (Rest) leg at different time-points before, and after single-legged leg presses (data adapted from Borgenvik. 2011)

As figure 3 finally goes to show, we see the "usual" increases in mTOR and p70S6K phosphorylation that are commonly held responsible for the downstream increases in protein synthesis, and which were obviously more pronounced in the exercised compared to the non-exercised leg. The latter may be ascribed to what the scientists cautiously label a ...

[...] tendency for BCAA supplementation to attenuate the elevation in the level of Rheb mRNA in both resting (1.7-fold under the placebo versus 1.2-fold in the BCAA condition) and exercising muscle (2.4-fold versus 1.5-fold).

This ameliorative effect on Rheb, the low-molecular weight GTPase located immediately up-stream of mTOR, in combination with the exercise induced reductions in REDD2 expression (another negative regulator of mTOR) the scientists observed in the exercised leg are actually where we are currently at, as far as our understanding of the complex protein synthetic machinery goes. It is here at the gene-level where amino acid supplementation and its effect on Rheb and exercise and its effect on REDD synergize and facilitate those muscle gains trainees have been making for years often without any understanding of the biological underpinnings.

And though we may eventually be able to squeeze out another 5-10% more muscle mass, when we eventually get the "whole picture", I seriously doubt that even the most thorough understanding of the underlying biomolecular processes will change such basic recommendations as "take your 25g of fast digesting whey as a bolus immediately post workout" (cf. "Never Sip Your Whey!") - or what would you say?

Whey Protein Alone Won't Cover the EAA Requirements of Hard Working Athletes, Study Says. Plus: US Whey More Digestible & 88% Higher in Leucine than Brazilian Whey

Not all protein supplements are created equal. And this goes for whey supplements from different countries, too.

In their accepted manuscript for LWT - Food Science and Technology, Cristine Couto Almeida and her colleagues write: "When the calculated AAS and PDCAAS based on the suggestion for adult athletes were considered, both [US & Brazilian whey protein] supplements exhibited suboptimal score values for several EAA [... and] were unable to supply the suggested adult athlete EAA requirement" (Almeida. 2014).

Shocked? I'd hope not. I mean, you don't even know what the scientists base their conclusion on - right? So before we even try to put things into perspective, it would be wise to take a look a the design of this in vitro study.

You can learn more about protein intake at the SuppVersity

Are You Protein Wheysting?

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High EAA protein for fat loss

Protein Timing DOES Matter!

Less Fat, More Muscle!

While the researchers from the Universidade Federal de Rio de Jaieiro acknowledge that whey protein, in general, is an effective adjunct to the diet of strength and even endurance athletes, they insist that there is too little "information regarding the WP supplement protein quality" and thus set out to "to investigate the protein quality of commercial WP supplements produced by U.S. and Brazilian companies based on in vitro digestibility (IVPD) assay, EAA, AAS and [protein digestibility-corrected amino acid] PDCAAS." (Almeida. 2014)

To this ends, the researchers acquired fifteen samples of whey protein (WP), soy protein, and caseinate isolate powder from a commercial retailer specialized on nutritional supplements. The supplements had been manufactured at different countries - eight from USA companies (WP-USA), and seven from Brazilian companies (WP-BRA). The supplements manufactured with soy protein and caseinate isolate powder were used as references in a study that yielded quite surprising results.

Figure 1: Essential amino acid composition of two commercial whey protein supplements (Almeida. 2014).

As you can see in Figure 1 the amino acid composition of the whey proteins from Brazil and the US varied significantly. The US whey, for example had significantly higher amounts of leucine, while the Brazilian whey was loaded with the essential amino acid lysine. While it is possible that the variations in the other amino acids are a result differences that were present in the milk, already, I would guess that the US whey was either openly (the scientists don't disclose the brands, otherwise I'd check) or secretly spiked with leucine to promote muscle anabolism.

Figure 2: Relative loss (%) of amino acids during simulated (in vitro) digestion in US and Brazilian whey (Almeida. 2014).

What are the numbers based on: Whether the amount of aminos is sufficient or not was calculated based on the WHO recommendation (WHO. 2007), assuming a normal (=comparatively low) protein intake.

If you consume twice the WHO suggestions for athletes, you are thus not at a risk of being deficient in any of the EAAs, but could maybe optimize the ratio of the individual amino acids by not covering your protein needs from a single protein source.

Even if we assume the latter was the case and the producer added free form amino acids to the whey, though, this does not explain the other differences, because if you add say 20g of leucine to 100g of EAA and measure the amino acid content of the 100g of your new mix, the content of all other amino acids would be lower.

 As you can easily see in Figure 1, though, this was not the case in the study at hand. Plus: There were also significant differences in the protein digestibility-corrected amino acid composition, i.e. the marker of whether or not the content of a certain essential amino acid per gram of protein was sufficient or not. In that, values <1.0 indicate there is too little of this amino acid in the mix.

Figure 3: Amino acid score and protein digestibility-corrected amino acid composition for the commercial
US and Brazilian whey supplements (Almeida. 2014).

As you can see in Figure 3, the latter was the case for threonine and valine in the US whey and for isoleucine and leucine in the whey protein from Brazil.

Figure 4: According to the standardized in vitro digestion assay (AOAC. 2012) the scientists used soy protein has by far the lowest digestibility and will thus be effectively delivering the lowest percentage of the amino acids it contains into your circulation (Almeida. 2014).

What does this mean? I must admit this sounds awful, but in practice it means only that you would end up getting your EAAs at an allegedly suboptimal ratio (I doubt we know what this ratio is, though) if you covered your complete protein needs with whey protein. In that, it is interesting that you would get too little threonine and valine form US wheys and too little leucine and isoleucine from Brazilian wheys.

Actual deficiency symptoms as you may have expected them, when you've read the statement that whey protein supplements were "unable to supply the suggested adult athlete EAA requirement" (Almeida. 2014), however are unlikely, because (a) I assume most of you won't live off whey protein as their only protein source and (b) even if you did, you would probably consume more than the WHO recommendation for athletes (WHO. 2014) that's at the heart of Almeida et al.'s calculation suggests | Comment of Facebook!

References:

• Almeida, Cristine Couto, et al. "In vitro digestibility of commercial whey protein supplements." LWT-Food Science and Technology (2014). 
• AOAC International, and George W. Latimer. Official Methods of analysis of AOAC International. AOAC International, 2012.
• Hsu, H. W., et al. "A multienzyme technique for estimating protein digestibility." Journal of Food Science 42.5 (1977): 1269-1273.
• WHO. "Protein and amino acid requirements in human nutrition." World Health Organization technical report series 935 (2007):

VPX Pre- & Post-Workout Nutrition Gets "Sponsored" Scientific Approval: +4% Lean Mass, -6% Body Fat, +13% Upper and +21% Lower Body Strength in 29 Days

Image 1: Supplemental double-whammy. VPX' now
"scientifically proven" pre- & postworkout products

This is one of those cases, where I cannot decide whether I should applaud VPX or just shake my head... the scientist in me says: "Hey, you know how that is - with a research grant from the government cutting edge science is impossible, especially if you want to investigate something as 'profane' as building muscle". The cynic skeptic, on the other hand, whispers: "Come on, what results would you expect, if the study was financed by the producer of the supplement under scrutiny?" I guess I will applaud skeptically and exercise special caution in my analysis of the latest study from the Department of Health and Performance at Baylor University (Willoughby. 2011).

As in previous studies (Willoughby. 2007; Willoughby. 2009), Darryn S. Willoughby and his colleagues availed themselves of a buckload of VPX supplements and recruited 19 previously recreationally active, yet untrained (*) men with an average age of 22.8 +/-4.67 years, a height of 179.5 +/-6.38 cm and a total body mass of 79.1 +/-16.13 kg for another study into the effects of two supplements, which are supposed to "advance you to the next level of fitness" (VPX. 2011). Strength and body composition (body fat measured reliably by DEXA, not body-impedance), venous blood sampling and muscle biopsies were performed on day 0 and day 29 of the 4-week study period, in the course of which the participants underwent a standardized resistance training protocol (upper-/lower-body split, 4x à week), which mirrored the one that had been used in Willoughby. 2009 already (*).

Figure 1: Illustration of the training regimen (based on Willoughby. 2011)

The bodybuilding-type beginner 2x split training regimen is unquestionably a huge plus of the study (cf. figure 1). Performed twice a weak, this is what real world training would look like and so that it stands out of question that the results of the study will translate into practice - at least for everyone who has not touched a dumbbell more than thrice a week within the last 12 and abstained from all sorts of performance enhancing supplements and drugs within the last 3 months (*).

The NO Shotgun approach to protein NO SyntheSize??? 

Figure 2: Ingredient profiles of
No Shotgun and No SyntheSize

More important than the identical training regimen was yet obviously the supplementation protocol, to which the participants were assigned in a double-blind randomization process (on a side note: "double-blind" means that not only the subjects, but the scientists, as well, did not know which participants received the placebo and which ones the VPX products). While half of the subjects consumed a maltodextrose placebo (27g pre, 27g post workout), the subjects in the "NOSS" group consumed the same amount of NO Shotgun and NO SyntheSize as their pre- and posworkout supplement, respectively. Now, as the names imply, both supplements are intended to increase nitric oxide production and protein synthesis, yet with a focus on the former in NO Shotgun that is loaden with arginine and a heap of stimulants and a focus on the latter in NO SyntheSize, the composition of which is pretty similar (cf. figure 2), yet without the "Redline Energy & Meltdown Fat Burning Technology" ;-)

Although there were no specifically dietary guidelines, the research did at least collect some nutritional data based on a 4-day questionnaire all participants had to answer at the beginning and end of the study. While there was a slight reduction in the total caloric intake in the carb group (interestingly mainly from carbohydates), neither the intra-group changes, nor the inter-group differences reached statistical significance.

More muscle, less fat! Trainee, what more can you ask for?

That there were no differences is yet something you cannot say of the changes in body composition the study participants underwent in the course of this 28-day intervention.

Figure 3: Relative changes (compared to baseline) in body composition after 16 strength training sessions in 28 days with either 54g of maltodextrin or 27g of NO Shotgun and 27g NO Synthesize pre- and postworkout (Willoughby. 2011)

As a passing view of the relative changes (compared to baseline) in figure 3 show, the NOSS group (receiving NO Shotgun prior and NO SyntheSize post workout) registered significantly more pronounced elevations in fat free mass (p<.023 indicates that the chances that this was sheer coincidence are 23%) and - contrary to the carbohydrate group - lost -6% of their body fat, while the carb eaters added another 2% of adipose tissue to their love-handles.

Figure 4: Changes in upper and lower body strength (in kg/kg body weight during bench press and leg press at 1RM) after 16 strength training sessions in 28 days with either 54g of maltodextrin or 27g of NO Shotgun and 27g of NO Synthesize pre- and postworkout (Willoughby. 2011)

Interestingly, the lean mass increase went hand in hand with likewise (statistically) significantly greater (p-values see figure 4) increases in both upper (+13% vs. +1%) and lower (+21% vs. +11%) strength in the subjects in the NO Shotgun + NO SyntheSize groups.

* you may have wondered what all the asterisks in the previous paragraphs meant... well, they indicate specificities in the study design detractors may call "precautions that ensure that the VPX supplements are sitting pretty" ... I mean the exact same supplementation protocol performed on a bunch of veteran bodybuilders would probably not have elicited any measurable effects on body composition - keep that in mind when you interpret the results.

Now, it obviously should not surprise you that the protein (and leucine) loaden and creatine, beta-alanine spiked workout supplements outperform simple sugar water. It is thus more interesting to take another look at the data from the 2009 "NO Shotgun only"-study, Willoughby et al. have done (Willoughby. 2009). On the exact same training protocol, yet with only 27g of NO Shotgun or placebo 30min preworkout, the participants lost less body fat (-1.21%), but gained the exact same ~4% of lean mass and comparable increases in bench press and leg press 1RM (+8.82% and +18.4%, respectively).

Scientifically proven ingredients make scientifically proven products

I leave it up to you whether or not the results of this study will influence your next supplement purchase - after all, even the VPX guys will be aware that their supplements are not so unique that intelligent people like you would not be able to identify the key ingredients in their products (EAAs, hydrolized protein, creatine, beta alanine, some workout-boosting stimulants, etc.) and realize that there are way more than those two products which would probably have produced identically (within statistical margins) results, if, and here we've come full circle, if their respective manufacturers had the money and the balls to do scientific studies on their products.