Pre Workout Protein Supplementation 101: Slow or Fast, Bolus or Pulse? Protein Synthetic Response is Identical!

Should she drink her protein shake all at once or in 33ml gulps every 15min, if she has it before her workout? And wait, wouldn't it be better to have the shake afterwards, anyway? A recent study provides some answers.

I don't know if you notices, but it has been a while since the last study from the Exercise Metabolism Research Group at the Department of Kinesiology of the McMaster University in Hamilton, Ontario, Canada, hit the SuppVersity news. Allegedly, I mentioned Stuart Phillips only a couple of days ago, when I referenced the West study on "Associations of exercise-induced hormone profiles and gains in strength" (West. 2012), in the context of the questionable significance of post-exercise increases in testosterone levels, as far as muscle and strength gains are concerned - for those who missed that, it was  in the "Anabolic Workouts Revisited" post from last Monday. That was however about it as far as the news of the last weeks are concerned. So I was already wondering, when the first of you would be showing initial signs of "acute protein synthesis in response to protein ingestion study withdrawal symptoms", when I hit onto the latest study Phillips' group at McMasters participated in. The lead author of the study is however Louise M. Burke who's currently working at the Australian Institute of Sport in Canberra, Australia.

What happens if you ingest your post-workout protein before the workout ;-) ?

The aim of the study, of which the authors explicitly state that it is a quasi-followup to previous results which have conclusively identified fast acting protein sources (mostly whey, in some instances EAAs) as superior triggers, or I should say, promoters of post-exercise protein synthesis, was

"[...] to investigate the effects of manipulating patterns of aminoacidemia from protein sources consumed before a bout of resistance exercise bout." (Burke. 2012)

To this end, Burke et al. simulated the ingestion of slow or fast protein sources by bolus vs. pulse feeding of a leucine-enriched (+5g) whey protein drink (Nestec by Nestlé, sponsor of the study; the reason for the enrichment was to make sure that there would be a decent amount of leucine in each pulse serving).

• Bolus (B) - 1 x 500ml w/ 25g whey + 5g leucine drink, 14 x 33ml placebo every 15 min
• Pulse (P) - 1 x 500ml placebo drink,  14 x 33ml w/ 1.79g whey + 0.36g leucine each
• Placebo - 1 x 500ml placebo drink, 14 x 33ml placebo drink

The subjects, 12 resistance-trained men (age: 27y; body mass: 94.3kg; 1 RM single leg ext.: 42.8kg) with greater than 2 yr of experience of regular (at least twice per week) strength training, who had followed a standardized diet before each of the testing sessions (energy content of 80kcal/kg BM; 45%/34%/21% of the energy from carbs/fats/protein), started consuming their large 500ml beverage and the subsequent 14 small 33ml servings (every 15 min) 45min before they performed a standardized leg training session:

"This bout consisted of a  standardized warm-up, followed by 10 sets of 8–10 repetitions of leg extension at a workload equivalent to 80% of the specific leg 1 RM with 2-min recovery between sets. The leg that performed exercise was alternated for each trial. The duration of the resistance bout was approximately 45 min." (Burke. 2012)

Before during and after the exercise bout blood samples were collected. Muscle biopsies from the vastus lateralis of the exercised leg were taken 45min before, as well as after 1 h and 5 h of recovery.

Figure 1: Serum leucine and insulin levels after bolus and pulse ingestion, expressed relative to placebo group (left) and fractional protein synthesis rate (in %/h) during the 5h following the rest period after the workout (right; based on Burke. 2012)

As you can see in figure 1 the results basically confirmed the scientists expectation that the provision of protein before a workout would work just as well, because - more than anything else - it is the availability (or should I say abundance?) of amino acids in the blood stream that is the main determined of post the actual fractional protein synthesis rate (FSR) after moderate  volume workout like the one the participants conducted in the study at hand. In addition it did, at least as far as the protein synthetic response goes, neither make a significant difference, whether the subjects had all their protion at once before the workout or consumed it in 15min intervals before and during the workout.

Bolus or pulse = fast or slow? Pulsed whey does not equal casein (imho)

What I am personally not happy with, though, is the way Burke et al. equate the "pulse" protocol to the ingestion of a "slow" protein source, such as a micellar casein protein, for example. While they put that into perspective in the discussion of the results (see below) and despite the fact that do see the rationale of Burke et al. not to use a real slow digesting protein like casein in order to have absolutely identical amino acid compositions and to exclude other confounding factors, it is at least in my humble opinion somewhat confusing for the "average" reader. And while it is likely that the results for a "real" slow digesting protein would be similar, this would warrant direct experimental evidence. A fact the authors only hint at indirectly towards the end of the discussion of their results, when they state:

"[...] A specific issue in interpreting the finding of these previous studies and in increasing the utility of the concept of ‘‘fast’’ and ‘‘slow’’ dietary proteins is the difficulty of determining the individual and interactive contributions of the different AA composition of protein-rich foods and the digestibility of proteins or protein-rich meals to the pattern of delivery of these AAs. Our protocol, in which the same (fast) protein was consumed to achieve its traditional AA profile or as a series of small divided feed-ings to replicate the plasma leucine response associated with a slow protein, provides an opportunity to differ-entiate these effects." (Burke. 2012)

But hey, who knows, maybe that's going to be the research question of the next paper... and in the mean time it will spare you to buy two or even more different protein powders ;-)

"So what's better Dr. Andro? Pre- or post-workout protein supplementation?"

I guess most of you won't care anyways, as there is another question that's now preying on your minds... but to be honest, I can't provide you with a definite answer to it (see headline). What I can do, however is compare the study by West et al. (West. 2011) which used 25g of whey protein post workout to the one at hand (Burke. 2012).

Figure 2: Fractional protein synthesis (FSR in %/h) after the workout with post workout protein ingestion (West. 2011) and pre workout protein ingestion (Burke. 2012) on the left; relative increase in FSR in the two trials (compared to fasted for West. 2011 vs. Placebo for Burke. 2012) on the right (please mind that this is by no means a scientifically valid comparison, it's more of a "food for thought illustration"!)

I've done just that for you in figure 2, and what this comparison tells you is that it does not make a difference, whether you ingest all your protein as a bolus after a workout or start "pulse ingesting" (mind my words in the previous paragraph wrt to "slow != pulse") your protein 45min before the workout - at least, if you take the relative increase in fractional protein synthesis as a measure (figure 2, right). 

Unfortunately, both the composition of the protein supplement (25g whey in West. 2011 vs. 25g whey + 5g leucine in Burke. 2012), as well as the exact outcome variables that were measured (1-3h and 3-5h FSR in West. 2011 vs. 5h post workout FSR in Burke. 2012) were different, so that the comparison of the relative increases in protein synthesis I plotted on the right hand side of figure 2 is actually not 100% valid.

Moreover, and this is something I know a couple of you will now be thinking of, this comparison does by no means allow for any quantitative predictions with respect to the question of ...

What would happen, when you do both: Pulse ingest before and bolus ingest afterwards?

If  you don't remember or - even worse ! - have not read the previous SuppVersity post "Protein Synthesis Beyond the '20g Limit': Study Shows Exercise Facilitates 32% Greater Increases in Fractional Protein Synthesis With 40g Instead of 20g of Whey PWO" click on the image and (re-)read it ;-)

In view of the fact that the existence of a "threshold level" of protein intake, where the addition of even more protein won't yield any further benefits appears to be self-evident, the exact amount of this limit has yet still to be determined. At least after a workout, it seems that this threshold would be higher than the 25g and 20g of protein the subjects ingested in the Burke and West studies, respectively (please read my previous post "Protein Synthesis Beyond the 20g Limit" for a more detailed discussion of this topic).

That being said, it is reasonable to assume, but would likewise warrant experimental verification, that the combination of both protocols could increase the fractional protein synthesis even further. Yet while I am 100% sure that they won't simply add up, I would hesitate to bet money that the difference would actually reach statistical significant... at least with a low volume leg extension workout as it was used in the studies at hand.

References:

• 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.
• West DW, Burd NA, Coffey VG, et al. Rapid aminoacidemia enhances protein synthesis and anabolic intramuscular signal-ling responses after resistance exercise. Am J Clin Nutr. 2011; 94:795–803. 
• West DW, Phillips SM. Associations of exercise-induced hormone profiles and gains in strength and hypertrophy in a large cohort after weight training. Eur J Appl Physiol. 2012 Jul;112(7):2693-702.  

Whey or Casein, Pulse or Spread Evenly Across the Day? Does it Even Make a Difference in Terms of Fat Loss and Lean Mass Retention on a Diet? New + Old Empirical Data!

Image 1: Instinctively right? Milk contains soluble (=whey) proteins and casein. Are we overthinking things, when we rip them apart and does it even make a difference? Or is timing all that counts?

It's funny "overthinking", right next to overtraining and overdieting, has become one of the most common problems among the health and fitness enthusiasts who spend equal (or even more) time online as in the gym. "Would it be better if I take my BCAAs at a 3:1:1 or 2:1:1 ratio?", "Does it matter if my protein powder is 10% hydrosolate, 50% isolate and 40% concentrate or has a 30/50/20 ratio?" All that may well make a difference, but let's be honest: Look at the things 80% of these people are eating day in and day out and the way they throw the weights around in the gym and contrast that to a question like "Will my post-workout protein synthesis be 5% greater, when I switch from concentrates to hydrosolates?" ... enough of the ranting, though. After all this post is actually about one of the more sensible among these world-shattering questions:

Q

Will it make a difference, whether I use casein or whey protein on a diet and... what's the significance of having my daily allotment of protein spread evenly across the day vs. mostly (80%) in one sitting, when I am dieting?

In order to find the answer to this question a group of French scientists recruited 41 healthy, but chubby subjects (BMI ~32kg/m²; age ~33y) and put them on a relatively moderate caloric deficit that was calculated based on their basal energy requirement (what you would need lying around all day). In all four arms of the study, the macro-nutrient composition (25% as proteins, 25% as lipids, and 50% as carbohydrates) and energy content per pound of lean body weight (average energy intake 5.87 MJ per day) of the meals, which were prepared according to personalized menus the subjects received from trained dietitians, were identical.

Figure 1: It did not make a difference if the protein was ingested either spread equally across the day or as a pulse mostly (80%) in one sitting (top), fat and weight loss after the 6 week study period were virtually identical (data based on Adechian. 2012)

The little information on the exact menu choices the scientists offers includes a list of stable foods, such as various proportions of spinaches, broccoli, lentils, or green beans, butter, bread, fruits, soy yogurt, rice cakes and gingerbread and suggest that we are dealing with the typical "your dietitian recommends diet", here. With one exception, of course, the main protein source of all four experimental diets were dairy proteins (~80g; >80% of total protein). Casein and whey aka "milk soluble protein"* (see red box above), which were to be ingested either spread equally across or in a "pulsed" fashion (see figure 1, left):

*Note: the scientists refer to whey as "milk soluble protein, I stuck to the terminology in the graphs, but in essence these are mainly β-lactoglobulin, α-lactalbumin, as well as serum albumin, immunoglobulins, lactoferrin, and other minor fractions and thus the same you would find in your average whey concentrate which is, as you may have notices "more soluble" than casein (cf. Lacroix. 2006)

• casein spread- subjects consumed ~20g of a casein protein supplement 4x a day
• milk spread - subjects consumed ~20g of milk protein supplement 4x a day
• casein pulse - subjects consumed the lions share, i.e. 80% of their ~80g of casein, as part of their 2nd meal, so that the protein intake over the day was 6.4g / 64g / 3.2g / 6.4g (see figure 1)
• milk pulse - same as above, but with milk instead of casein protein

In view of the overemphasisze nutrient timing has gotten as of late withing the physical culture and the assumption that you would expect to see profound differences based on when you consume how much of fast or slow, high (milk) or average (casein) leucine protein etc., it may be disappointing that the weight loss was absolutely identical in all four arms of the study (-7.5 ± 0.4 kg).

Differences are few and far between: Weight loss, fat loss, muscle loss - NOT different! 

What may yet surprise even you, a seasons SuppVersity veteran, who will probably already have expected the non-significant (in fact non-existent) differences in terms of weight loss, could be surprised that the changes in body composition (see figure 1, bottom), i.e. -5.1 ± 0.2 kg reduction in body fat mass and -2.2 ± 0.2 kg reduction in lean body mass, were identical.

Since the same goes for the changes in the fat "liberating" proteins lipoprotein lipase (LPL) and adipose triacylglycerol lipase (AGTL), the fat "forming" protein fatty acid synthase (FAS), and three of the usual subjects, i.e. leptin, the adipoQ gene which is responsible for encoding adiponectin, of which recent research suggests it may be even more important than leptin for your metabolic health (Li. 2012; Hickman. 2012), and the reduction in the pro-inflammatory monocyte chemotactic protein-1  (MPC-1), the slightly more pronounced meal-induced postprandial protein synthetic response in the casein group at the end of the study period is actually the only difference based on which you could argue for one over the other protein source:

Figure 2: While the changes in LPL, AGTL, FAS, leptin, AdipoQ and MCP expression were identical (left); the post 6-week protein synthetic response to identical meals was slightly more pronounced in the casein group (right), the overall significance of this finding is yet questionable in view of identical lean mass losses - it could yet become important on a diet + exercise regimen as in the Demling study discussed in the bottom line box  (data based on Adechian. 2012).

Whether the measurable advantage of casein during this test (the evaluation was carried out by leucine tracer infusion, by the way) is just an experimental artifact or

Adherence is the key to success: While there was no difference in terms of the hunger the subjects felt when they were on the diet, the fact that only 23 of the initially 41 subjects did make it through the 6- week on ~ 1,500kcal/day is quite telling, also in view of the perceived inability to lose weight - if you can't stick to a by no means crazy caloric restriction for 6 weeks, how can you expect to get lean and stay lean, when the inevitable prerequisite for the latter is that you totally revamp your dietary habits for the rest of your life not just six, eight, or twelve weeks.

• maybe something like "leucine resistance" in response to the higher leucine concentrations after the ingestion of the milk protein supplement in the course of the study period, or
• alternatively, the greater IGF-1 response to casein (cf. Hoppe. 2009, a study which compares whey vs. casein, but would obviously suggest an advantage of casein over milk = whey + casein, as well); unfortunately IGF-1 wasn't measured, but the insulin levels which were minimally higher in the casein group could support that hypothesis,

... is questionable. Since the same is true for the practical relevance of the ~10-13% larger leucine balance during the postprandial phase of the post-diet whole body protein metabolism test in week 6, I would not fret about this difference too much, though.

Maybe, just maybe, the adipocyte morphology could make a difference

What I would consider significant, though it did not reach that status (probably due to the low number of participant that actually made it to the end of the study, see red box on the right), is the slight but in my eyes potentially important superiority of the equally spread protein ingestion in terms with respect to the before vs. after adipocyte diameter in the casein group:

Figure 3: The difference did not reach statistical significance, but if we take for granted that greater reductions in adopcyte sizes are associated with healthier metabolic profiles, you would be better advised to take your casein protein equally spaced across (15% reduction in adipocyte size vs. 7%, only, for pulsed casein intake) the day... for whey, aka "milk soluble protein", on the other hand it does not seem to matter (data calculated base on Adechian. 2012)

Now, even if we assume that this made a difference and a greater reduction in adipocyte size was a significant advantage, which it probably is from a health perspective, as Skurk et al. state that there is

"[...] a differential expression of pro- and antiinflammatory factors with increasing adipocyte size resulting in a shift toward dominance of proinflammatory adipokines largely as a result of a dysregulation of hypertrophic, very large cells." (Skurk. 2006)

and a recently conducted human trial, by Rizkalla et al. the main message this study should be sending out is not that it does not make a difference whether you use casein or milk protein as your main protein source on a diet, but that a high protein diet with a mediocre caloric reduction of ~20-25% and supplemented with high quality dairy protein (whey or casein) works: After all, more than -1kg of weight loss per week, 68% of the weight loss from fat in the absence of exercise is more than your average celebrity XYZ diet will do for you ;-)

Whey or casein? It's high cysteine content that can help to replenish your glutathione (=the master antioxidant) pools would be another factor that speaks in favor of whey. Whether normal-weight individuals on an already optimized dietary regimen would benefit to the same extend as the obese young men in the 6-week whey supplementation trial, Vatani et al. describe in the August issue of Appetite, is however questionable. After all, the increases in HDL the total antioxidant capacity and glutathione is as questionable as any possible negative influence of the starchy placebo the researchers used in that study (some of you may have seen the link on the SuppVersity Facebook Wall, already).

Figure 4: Fat loss and lean mass gains in formerly overweight police officers after 12 weeks of training and dieting with or without casein / whey hydrosolate (Demling. 2000)

Moreover, one of the few long-term (=non acute protein synthesis) studies investigating the differential effects of concomitant whey vs. casein hydrosolate protein supplementation, found statistically significant higher body fat reductions and lean mass gains in those 33-34 year-old police officers who supplemented their 12-week diet + strength training regimen with 2x37g of casein hydrosolate (8h apart; for the exact data see figure 4; Demling. 2000).

Note: since both the whey (Pro-Score Champion Nutrition) and the casein protein (MET-Rx USA) in this study were hydrosolates the differences in lean mass gains and fat loss are depend primarily on the amino acid composition of the proteins, and not, as it would be with micelle casein vs. whey, the absorption kinetics!

Bottom Line: Against that background the study at hand supports previous findings of the importance of a threshold intake of protein. Interestingly, it did not confirm the notion that this threshold intake should be spread equally across the day, which is something most commenters (me included) read into the seminal paper by Loenneke et al., which found a statistically significant negative correlation not between total protein intake, but between the number of meals with 10g or more essential amino acids in them and abdominal obesity (Loenneke. 2012). So, does timing matter, or does it not? 

1. It does matter, when you work out, there is ample evidence to support that the ingestion of protein in the vicinity of the workout cannot just amplify the protein synthetic response but will also results in an increase in real world muscle gains.
2. It appears that it does not matter, when you are dieting (only), though; not just the study at hand, but also the success many people report on intermittent fasting regimen, would support the notion that the more sustained anabolism you may be able to achieve by ingesting say 4x25g of protein instead of 1x80 + 2x10g has, compared to the total amount of protein you eat, relatively little influence on the conservation of lean body mass, when you are dieting.

And as far as the choice between casein and milk soluble protein, aka whey (see first red box), is concerned (see box on the right, as well), it would appear prudent to assume that a combination of both - just like nature intended it - would be the best choice as a "standalone" protein source (cf. "Whey and Casein Work Hand in Hand for Protein Anabolism, but Scientists Overlook Fat, When They Reassemble Milk"), while the higher leucine content and faster digestibility render whey the better candidate for classic "supplementation", as in having an additional shake before you start preparing your whole-foods post-workout meal, which should - and I hope it's not really necessary that I say that - obviously include a significant amount of protein (fish, eggs, meats, and if you will even more dairy ;-), as well. The usefulness (again, not necessarily the superiority!)  of slow digesting protein is something you should be aware of, anyway, right? If not re-read the "3.2kg of Lean Mass Over Night W/ 40g of Slow Digesting Protein 30min Before Bed!?" post from February 22, 2012.

References:

• Adechian S, Balage M, Remond D, Migné C, Quignard-Boulange A, Marset-Baglieri A, Rousset S, Boirie Y, Gaudichon C, Dardevet D, Mosoni L. Protein feeding pattern, casein feeding or milk soluble protein feeding did not change the evolution of body composition during a short-term weight loss program. Am J Physiol Endocrinol Metab. 2012 Aug 14.
• Demling RH, DeSanti L. Effect of a hypocaloric diet, increased protein intake and resistance training on lean mass gains and fat mass loss in overweight police officers. Ann Nutr Metab. 2000;44(1):21-9.
• Hickman IJ, Whitehead JP. Structure, signalling and physiologic role of adiponectin - dietary and exercise-related variations. Curr Med Chem. 2012 Aug 9.
• Hoppe C, Mølgaard C, Dalum C, Vaag A, Michaelsen KF. Differential effects of casein versus whey on fasting plasma levels of insulin, IGF-1 and IGF-1/IGFBP-3: results from a randomized 7-day supplementation study in prepubertal boys. Eur J Clin Nutr. 2009 Sep;63(9):1076-83. 
• Lacroix M, Bos C, Léonil J, Airinei G, Luengo C, Daré S, Benamouzig R, Fouillet H, Fauquant J, Tomé D, Gaudichon C. Compared with casein or total milk protein, digestion of milk soluble proteins is too rapid to sustain the anabolic postprandial amino acid requirement. Am J Clin Nutr. 2006 Nov;84(5):1070-9.
• Li FY, Lam KS, Xu A. Therapeutic perspectives for adiponectin: an update. Curr Med Chem. 2012 Aug 9.
• 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. 
• Rizkalla SW, Prifti E, Cotillard A, Pelloux V, Rouault C, Allouche R, Laromiguière M, Kong L, Darakhshan F, Massiera F, Clement K. Differential effects of macronutrient content in 2 energy-restricted diets on cardiovascular risk factors and adipose tissue cell size in moderately obese individuals: a randomized controlled trial. Am J Clin Nutr. 2012 Jan;95(1):49-63.
• Skurk T, Alberti-Huber C, Herder C, Hauner H. Relationship between adipocyte size and adipokine expression and secretion. J Clin Endocrinol Metab. 2007 Mar;92(3):1023-33.
• Vatani DS, Golzar FA. Changes in Antioxidant Status and Cardiovascular Risk Factors of Overweight Young Men after Six Weeks Supplementation of Whey Protein Isolate and Resistance Training. Appetite. 2012 Aug 10.