I saw this abstract in my Facebook feed, and when I saw, “randomized controlled trial” in the title, I knew I had to read it because good randomized controlled trials in fitness and nutrition are hard to come by. Let’s say that I was pleasantly surprised and excited by this one; and truthfully, that doesn’t happen that often.

Before we talk about the study, I think it’s important to differentiate between “high-protein” diets and “low-something else” diets. “High protein” means that the first maconutrient of importance is protein and when figuring how much carb and fat to eat, the calories not taken up by protein are then taken up by the other two macronutrients. “Low something-else” means that the first macronutrient taken into consideration is the “something else” (these days it’s usually carbohydrate), and then the rest is available for fat and protein. This doesn’t mean that a low-carb diet is automatically a “high protein” one, or, more importantly, that a “high protein” diet is automatically a low-carb one.

Additionally, it’s important to note that the typical bodybuilding definition of “adequate protein” (1g per pound of body weight, or 2.2g per kilogram of body weight) is not the research definition of “high protein” and that this definition changes from study to study.

One of the concerns of being in an energy deficit is loss of muscle mass. Energy deficits, however, aren’t restricted to just fat-loss plans; they can happen inadvertently to athletes or professions where increased activity and/or not-quite-enough-food scenarios like military operations are not uncommon. So what happens if we play with the protein intake of people when they’re in an energy deficit? Can eating more protein cause less loss of muscle mass?

Pasiakos SM, Cao JJ, Margolis LM et al. Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial. FASEB journal, 27, DOI: 10.1096/fj.13-230227, 2013

Methods:

Target population: Subjects were volunteers who were physically active at least 3-4 days per week, weight-stable for at least 2 months, with a BMI between 22-29 and a VO2(peak) between 40-60ml/kig/min (described as recreationally fit.)

Subjects were admitted to a metabolic ward. This is basically a controlled environment where no outside food is permitted. Subjects were required to be in their rooms with lights out at 11pm. No supplements, alcohol, smoking or medications were allowed. Only medications provided by the investigators were allowed (usually acetominophen.) This was a serious experiment (yay!)

Subjects were randomly assigned to one of three groups (they actually used the term “randomized block design” which made me so happy to see–finally): RDA (0.8g/kg body weight or 0.4g/lb), 2xRDA (1.6g/kg body weight, or 0.7g/lb) or 3xRDA (2.4g/kg body weight, or 1.1g/lb.)

For the first 10 days of the experiment, the goal was to stay at a consistent weight. Maintenance calories were calculated from a variety of sources. After accounting for the calories provided by protein, less than or equal to 30% of calories were allotted to fats and the remainder to carbohydrates. Everyone got a multivitamin. All meals were prepared in the kitchen of the metabolic ward and meals were supervised to ensure consumption (clean your plate!)

After the weight-maintenance period, calories were cut by 30% and physical activity was increased 10% above the total daily energy expenditure. Protein intake remained the same as per each subject’s assigned group.

In terms of exercise, subjects all performed treadmill and stationary bike sessions daily at a low-to-moderate intensity, as calculated by their pre-study VO2(peak) and monitored with portable heart rate monitors. Subjects also performed low-intensity, low-volume resistance training (3 sets of 15 reps per muscle group, ony single-joint movements–no compound lifts) using workloads tested in the pre-study period. Frequency, intensity, mode and volume were kept constant throughout the study.

On day 12 (2 days after 30% of calories had been cut), the treadmill and stationary bike sessions got longer to produce a 10% increase in caloric expenditure to create an overall energy deficit of 40%. Each group burned approximately 480-490ish calories per day with exercise.

Measurements:

Subjects were weighed every day after an overnight fast (i.e. first thing in the morning) and after they had voided. The authors wrote, “…to ensure either weight maintenance or weight loss…” in this section, and added that weight loss occurred during the weight-maintenance period (days 0-10), carbs were added to the diet; but didn’t mention what happened if weight loss wasn’t occurring (maybe that didn’t happen?) DEXA was used for body composition measurement on day 9 and day 30.

A stable isotope infusion protocol with muscle biopsies was used on day 10 and 31 of the experiment to examine protein synthesis as well as cell signalling and gene expression. Subjects did not exercise for 2 days prior to each testing day. Muscle biopsies were taken at time 0, 120 minutes, 300 minutes and 480 minutes after the phenylalanine infusion was started. Right after the 300 minute biopsy was taken, subjects were given a Boost drink (as in the Nestle product; it’s not some weird science-y term for anything) with labelled phenylalanine to keep the protein entry rate the same. A Boost drink contains about 20g of protein.

Nitrogen balance was calculated from the amount of nitrogen consumed in the diet and subtracting calculated losses in feces and miscellaneous losses as well as a 24h urine collection. This was performed on days 9, 14, 17, 24 and 30 (I’m not quite sure why day 17)

Statistics

The authors based the sample size estimation on an 85% power, but didn’t mention what effect size they had based it on. This is a little problematic, as we’re not given any idea what difference they thought would be important. They estimated needing 12 subjects per group.

Subjects were stratified by gender, BMI (22-25 vs 25.1-29), VO2(peak) and pre-study dietary protein intake (0.8-1.6g/kg vs 1.6-2.4g/kg). I’m a little surprised they went with so many levels of stratification, as it means that they would be unable to analyse the effects of pre-study BMI and pre-study protein intake on their outcomes.

They used variations of ANOVA’s for statistical comparison and stated the use of a Bonferoni correction for multiple tests of significance (Holy crap, I don’t think I’ve seen that in a nutritional study yet!)

[I don’t know why, but all the results are reported as means with standard errors, and not standard deviations. It’s REALLY ANNOYING, since I have to do math for every single standard error. Standard error is for POPULATION estimates, not sample estimates, people.]

Results

I can sense this post is getting long in the tooth, so I’m going to stick to the major points, and not review every single result.

Overall, everyone lost weight.

Over the 10-day weight-maintenance period, no one lost weight (which is the way it was supposed to be.) Over the 21-day energy-deficit period, the RDA group lost an average of 3.5kg (SD 1.1), the 2xRDA group lost an average of 2.7kg (SD=0.7), and the 3xRDA group lost an average of 3.3kg (SD=1). Statistically different? Yes. Important? I really don’t think so.

In terms of fat loss, the RDA group lost an average of 1.3% body fat (SD=1.1%), the 2XRDA group lost an average of 1.8% body fat (SD=1.5%), and the 3XRDA group lost an average of 1.9% (SD=1%).

The crux of this paper is in the percentage of weight loss attributed to fat loss and to fat-free mass.

The authors report an average percent of weight loss from fat loss as 41.8% in the RDA group, vs 70.1% and 63.6% in the 2XRDA and 3XRDA group respectively. However, in terms of absolute mass of fat loss, the numbers were 1.6kg (SD=0.7) for the RDA group, 1.9kg (SD=1.1) in the 2XRDA group and 1.9kg (SD=0.7) in the 3XRDA group.

For fat-free mass loss, the RDA group lost an average of 2.3kg (SD=1.1), the 2XRDA group lost an average of 0.8kg (SD=0.7) and the 3XRDA group, 1.2kg (SD=1).

I’m leery of getting too deep into protein synthesis results because I feel that the assumption that changes that occur in phenylalanine infusion studies don’t necessarily reflect changes in the size of the muscle itself. Protein acrection=muscle growth seems like an overly simplistic way to look at the forest through one tree.

Nevertheless, the increase in protein synthesis that is normally observed after consuming protein (with, or without exercise; in this study, without exercise for 2 days prior to testing), was observed in all groups in both the weight-maintenance phase and the energy-deficit phase. The authors state that in the RDA group, that the protein synthetic rate was blunted during the energy-deficit phase, but we’re talking about the difference of 0.01% and -0.001%. The sign difference makes the statistically test significant, but again, I’m not so certain that it’s _important_.

I’m skipping the gene expression and nitrogen balance stuff, because its importance is vanishingly small in this study. If you can think of a salient argument as to why it should be discussed here, I’m all ears.

Discussion

I always have problems with statistical testing on percentages as well as absolute numbers, particularly when they might not agree. I think we can agree that average weight losses of 3.5kg vs 2.7kg vs 3.3kg are not really meaningfully different from one another (even taking the standard deviations into consideration.) That’s a difference of almost 2 pounds between the lowest and highest group. And an average fat-loss of 1.6kg vs 1.9kg vs 1.9kg is even less of a between-group difference. So when the percentage of weight-loss attributable to fat comes up statistically significantly different (42% vs 70% vs 64%), I’m really not convinced that it’s any more important than it was when we were talking 1.6kg vs. 1.9kg vs 1.9kg. So, do I think, after reading this study, you lose more fat on a higher than RDA diet? No.

What DOES stick out in this paper is the loss of fat-free mass. You should definitely NOT interpret fat-free mass as muscle loss, as it includes all non-fat tissues including bone, and organs as well as muscle. However, on the whole, I think most of us would like to minimize fat-free loss when losing weight, regardless of where it’s coming from. I don’t think we can dismiss the average fat-free loss of 2.3kg vs. 0.8kg vs. 1.2kg as being trivial. While it is only 3.3lbs at the largest difference, a greater than 2-fold difference is nothing to sneeze at. While weight-loss (fat-free or otherwise) is definitely not a linear process, when you start adding up the fat-free losses over time (even assuming that the losses get smaller and smaller as time goes on, since it’s highly unlikely to be linear), you’re still going to end up with very different numbers between RDA and higher-than-RDA protein groups.

What’s also shown in this study is that consuming protein higher than 2xRDA doesn’t seem to have any additional benefit.

The main limitation to this paper is whether we would still see fat-free preservation in the RDA group if their resistance training was allowed to be progressive; or conversely, whether we would see fat-free mass _gains_ in the higher protein group. However, this paper does give a compelling reason to eat at a higher-than-RDA amount of protein per day, as it successfully isolates the contribution of diet in fat-free mass preservation. What we don’t know is whether adding in progressive resistance training has a confounding or interacting effect that might change the picture entirely.

So who does this research benefit in the end?

When we look at the sample: Volunteers who were physically active at least 3-4 days per week, weight-stable for at least 2 months, with a BMI between 22-29 and recreationally fit, the generalizability of this study has the potential to be quite wide. A BMI of 25-29 would be considered overweight. If you’re 5’10” and between 175 and 205lbs, and feel like you have some fat to lose, this is probably useful information, and good justification to eat at 2xRDA, which is 0.7g of protein per pound of body weight–still nowhere close to the conventional recommendation of 1g protein per pound of body weight. In fact, 3XRDA, which doesn’t seem to provide any additional benefit is closest to the conventional recommendation, at 1.1g of protein per pound of body weight.

The other issue that isn’t quite clear in this paper is whether the protein requirement (which is based off body weight) slides DOWN as body weight decreases. If you started at 205lbs, which would be a protein requirement of 144g of protein (for 2XRDA), and you lost the reported average of 6 pounds in 30 days, would your protein requirement then become 140g? According to the authors, “Dietary protein intake remained constant during weight maintenance and energy deficit…” which suggests your protein requirement would stay at 144g of protein (at least for 30 days); but for how long? Forever? What happens when you reach a BMI in the ‘not overweight’ category at 175lbs, which according to formula would be 122g of protein? And how do these results vary for different starting points? I have a hard time understanding how the protein requirements of an individual trying to lose weight are different if they start off heavier or lighter, if they both start off with about the same percentage of mass as fat.

Increasing your protein intake higher than the RDA doesn’t appear to affect whether you lose more fat, but rather, how much non-fat mass you get to keep during periods of energy deficit. However, the 1g per pound of body weight rule doesn’t seem to apply as this study did not demonstrate any additional benefit past the 0.7g per body weight requirement. I don’t see much harm coming from consuming 1g of protein per pound of body weight (other than potential pocket-book damage), but I wouldn’t freak out if I wasn’t getting that either. More is better only up to a certain point.