Protein Timing Does Matter! Yet Only in Trained Men. More Than 2x Higher Relative Protein Retention W/ Immediate vs. 6h Post Whey Consumption in Bodybuilders vs. Rookies

Drop the weights, grab the shake! Timing matters for advanced trainees.
I guess you'll all have followed my suggestion to read Brad Schoenfeld's, Alan Aragon's and James Krieger's excellent review of the effects or protein timing on skeletal muscle hypertrophy, last year. In said paper, the two conclude that their review would "refute the commonly held belief that the timing of protein intake in and around a training session is critical to muscular adaptations" (Schoenfeld. 2013)

Certainly a reasonable conclusion based on the evidence they present. With the recent publication of a study by Hiroyasu Mori from the Department of Nutrition Management at the Hyogo University, future reviews will yet probably have to distinguish according to the training status of the athletes.
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In his latest study, Mori investigated the effect of the timing of protein and carbohydrate intake after resistance exercise on nitrogen balance in trained and untrained young men. By dividing his 20 healthy male subjects between the age of 20 and 29 into two groups, i.e.
  • those Mori calls "bodybuilders" and who regularly performed resistance exercise were assigned to the trained group (n = 10; mean age, 23 ± 4 years; height, 173.8 ± 3.1 cm; weight, 72.3 ± 4.3 kg) and
  • those of which Mori writes that they were "recreationally trained" but without resistance training experience were assigned to the untrained group (n = 10; mean age, 23 ± 1 years; height, 171.8 ± 5.0 cm; w eight, 64.5 ± 5.0  kg),  respectively,
Mori added an element to the equation that has been overlooked in previous studies. An element that would allow him to answer the question:

Do the same rules apply for untrained subjects and bodybuilders with 6.2 ± 2.8  years of training experience?

In view of the fact that the majority of studies that investigate the long(er) term hypertophy effects of resistance training are conducted with training noobs to make sure you can measure meaningful and statistically significant changes after only a few weeks, the previously cited conclusion by Schoenfeld et al. is also based mostly on data from rookies or the infamous "recreationally active" study participants.
Figure 1: Overview of the experimental design (Mori. 2014)
As you can see in Figure 1, both groups, i.e. the bodybuilders and recreationally active rookies, were subjected to the same exercise + supplementation protocol in this 4-week randomized crossover trial.
"In the P0 experimental period, subjects consumed protein and carbohydrate supplements 5 min after resistance exercise, and in the P6 experimental period, subjects consumed the same supplements 6 h after exercise. A washout period >7 days was applied before each experimental period. During each 11-day experimental period, the first 8 days were defined as an adaptation period for muscle to adapt to the energy and nutrients from the experimental food and supplements prepared by the examiner. During the next 3-day period (day 9 to day 11), 24-h urine samples were collected. The two experimental schedules are shown in Figure 1. " (Mori. 2014)
Because at least 7 days of adaptation and 3 days of urine collection are needed to calculate nitrogen balance (Jordan. 2010), the resistance exercise schedule in the P0 and P6 experiments lasted for 11 days: 8 days of adaptation (two cycles of resistance exercise for 3 days and rest for 1 day) and 3 days of urine collection.
Are urine collections valid measures of protein retention? I would prefer a 12-week study that measures the net muscle gain in response to immediate vs. 6h post supplementation as well. In the end, the 3+ day urine collection is probably still a better measure of the amount of protein that's actually used to "build lean mass" (remember this way we cannot distinguish where the protein was stored - we only know that is was not metabolized and excreted!) than the acute measures of protein anabolic signalling you see in many other studies - a measure of which Mitchell et al. have shown that it does not correlate with resistance training-induced muscle hypertrophy in young men (Mitchell. 2014), only recently.
Prior to each experimental period, body composition and one-repetition maximum (1RM) were measured, and questionnaires on daily activity were completed.
 "In daily experimental sessions, subjects performed the following resistance exercises: 4 sets of 8 to 10 repetitions of resistance exercise consisting of leg press, leg extension, and leg curl on experimental days 1, 5, and 9; bench press, shoulder press, and triceps pushdown on experimental days 2, 6, and 10; and lat pulldown, biceps curl, and rowing on experimental days 3, 7, and 11. All exercises were performed at 80% RM, and each set was followed by a 2-min break." (Mori. 2014)
Before the experimental session each day, subjects used a cycle ergometer (Aerobike 800; Combi Wellness Corporation, Tokyo, Japan) at 100 W for 10 min to warmup. Each exercise session was scheduled to take place between 10:00 and 11:00. The subjects were instructed not to participate in any other sports activities during the experimental period.

The diets were standardized to 1.5g/kg body weight

In spite of the fact that the bodybuilder group consumed ~13% more energy per day, both the total (1.5g/kg per day), as well as the supplemental protein intake (0.5g/kg of whey protein + 0.8 g/kg dextrin body weight) were standardized. In that, the amount of protein per meal was defined as follows:
  • P0 experimental period: Subjects had to ingest protein (0.3 g/kg body weight) and carbohydrate (0.8 g/kg body weight) immediately after resistance training.
  • P6 experimental period: Subjects had to ingest protein (0.3 g/kg body weight) and carbohydrate (0.8 g/kg body weight) 6 h after resistance exercise session.
The total energy intake, total protein intake, and protein intake per  body  weight  (kg) were  calculated  by  a  registered dietitian and the protein balance was assessed by analyzing the nitrogen excretion in the urine.
Figure 2: Net protein balance per body weight and per lean body mass (LBM) when the post-workout protein + carbohydrate shake was consumed immediately after or 6h after the workout (Mori. 2014)
And, as you can see in Figure 2, a comparison of the net protein balance in the two groups during the immediate post vs. 6h post consumption periods clearly indicates that protein timing does matter, even if it's just for the experienced resistance trainee, for whom the study at hand shows that his nitrogen balance is less positive than that of the rookie anyway.

Apropos total protein intake and nitrogen balance, the average total protein intake in the studies reviewed by Schoenfeld et al. was after all slightly higher (1.66 g/kg/day). This, as well as the general believe that bodybuilders should consume tons of protein make me question whether the results would have been different, if total protein intake had been 2.0 or even 2.5g/kg per day instead of just 1.5g/kg. Personally, I don't thinks so, but it would be worth a try, anyway.
(1) Thou shalt not wait 6h to consume protein after a workout (2) Thou shalt not wait 6h to consume carbs (not necessarily ultra fast digesting, though) after a workout either | learn why
Bottom line: If you're striving for maximal muscle and performance gains, specificity is key; and the study at hand specifies that you have to specifically make sure to get your post-workout nutrition "immediately" post workout and not 6h later if you (a) want to maximize net protein retention and (b) are already beyond those first happy months in the course of which you just have to look at a dumb- or barbell to grow ;-)

Ah, and when you're at it, I suggest you also include 5g of creatine monohydrate in your postworkout shake. The latter has after all also been shown to work a tad better, when it's consumed after the workout | learn more.
  • Jordan, Leora Y., et al. "Nitrogen balance in older individuals in energy balance depends on timing of protein intake." The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 65.10 (2010): 1068-1076.
  • Mitchell, Cameron J., et al. "Acute Post-Exercise Myofibrillar Protein Synthesis Is Not Correlated with Resistance Training-Induced Muscle Hypertrophy in Young Men." PloS one 9.2 (2014): e89431.
  • Mori, Hiroyasu. "Effect of timing of protein and carbohydrate intake after resistance exercise on nitrogen balance in trained and untrained young men." Journal of Physiological Anthropology 33 (2014): 24.
  • Schoenfeld, Brad Jon, Alan Albert Aragon, and James W. Krieger. "The effect of protein timing on muscle strength and hypertrophy: a meta-analysis." Journal of the International Society of Sports Nutrition 10.1 (2013): 53.
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