Controlled studies answers questions like "I'm on a 2000 calorie diet. How will fat loss be impacted if I eat most of those calories in the later part of the day versus the earlier part of the day?" That's what interesting to us, so let's look into this now.

Early Meal Patterns vs Late Meal Patterns: Controlled Studies


In all of these studies, calories were controlled and fixed for all groups. The only variable that differed was the temporal daily distribution of calorie intake. In late meal patterns, 67-100% of total daily caloric intake was eaten between 6 PM and bed time, and this was compared against an early meal pattern with an opposite pattern.

Starting with the earliest study and working myself down to the latest study, I'll briefly summarize the results, comment on the validity of the study, and interject whatever else of interest I find in each study.

Note that I will not include studies on Ramadan fasting. In loosely controlled studies on Ramadan fasting, fat loss and improvements in health markers is commonly found. This is a paradoxical and interesting finding, simply for the fact that people eat in the middle of the night, shortly before bedtime, along with a concomitant increase in intake of sugary treats and baked goods (and sometimes total calorie intake). However, these are rarely calorie-controlled studies, i.e. participants do not have strict guidelines about what they should eat, which is why I will not include them in this review.


Study #1


Study #4



Study #5

Since most people's eyes glaze over when someone starts talking cell biology, imagine your cell as an avocado. Picture the avocado with myriads of pins buried in it.

This is your cell. The pins are membrane proteins, some of which are nutrient transporters for glucose, amino acids, and so forth. The dark skin and the edible green flesh is the plasma membrane, which consists of two thin layers made of fatty acids. N-3 supplementation has the potential of altering this part of the cell. Generally speaking, plasma membranes with a higher % EPA and DHA enrichment are healthier; they have the potential of modulating signals in between the white pins/plasma proteins and the avocado core. The "avocado core" is the cell interior, which react in response to signals from the cell exterior.

Pins: are called integral membrane proteins."Integral membrane protein" is a catch-all term for proteins that shuttle nutrients or signals to the cell itself, or the avocado core if you will. Included in these are glucose transporter type 4 (GLUT4) and amino acid transporters, for example. Pretend these pins had the ability to move up and down through the skin and flesh of the avocado. The word for this is translocation, or protein targeting depending on how fancy you want to get.
Skin and edible part: this is the cell membrane, also called the lipid bilayer, or the phospolipid bilayer. A large part of these layers consists of fatty acids and their structure is highly dependent on the fat composition of your diet. This is where n-3 fatty acids exert their effect; by their incorporation into lipid bilayers, n-3 acids can modulate signals between the pins to the cell itself. In particular, it is the omega-3 series members docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) that facilitates these processes. When I talk about "n-3 supplementation/intake", it is often in specific reference to EPA and DHA, and not ALA.

Avocado core: cell interior, where transcription (i.e. muscle protein synthesis) takes place based on feedback from the cell exterior and pins.

Obviously, this is a vastly simplified version of the cell, but I've found the avocado analogy somewhat helpful in dumbing down the cell and its workings.

The fact that n-3 is incorporated into the plasma membrane of all your cells - be it brain cells, muscle cells, and so forth - explains how these fatty acid can have so diverse effects on human physiology and health. Let's take a look at what they do.

Dietary epidemiology have also shown a link between n-3 and mentally debilitating disorders such as Alzheimer's and depression. Furthermore, n-3 may affect the development of many facets tied to brain function, such as intelligence, vision and mood. For example, infants who do not get enough n-3 from their mothers during pregnancy might be at risk for developing vision and nerve problems.
Other epidemiological trials have suggested a protective effect of n-3 on cancer and tumour growth; in particular, prostate, breast and colon cancer.


Omega-3 Fatty Acids: Effects on Body Composition


What about n-3 and fat loss? I have not seen a single study which shows that adding n-3 alone will generate fat loss in lean subjects, but overweight/obese subjects sometimes see a small - but clinically significant - effect of n-3 supplementation. There are some exceptions to this, i.e. some studies do show a clinically significant fat loss effect in lean subjects, but the sample size and diet design is extremely poor - such as in this widely cited study by Couet, et al.
However, I'll note that it's a profound flaw to look at the effects of n-3 and fat loss in lean subjects and conclude that there is no benefit. Why? Because the effect of n-3 on fat metabolism and adipose tissue is not acute. It takes approximately 4-6 weeks of n-3 supplementation to change the fatty acid composition of the cell membrane, and controlled interventions on n-3 supplementation and fat loss have never been longer than 12 weeks in duration. In the case of Brilla, et al., who looked at fat loss in lean subjects, it was 10 weeks.
The implication of this is that the study, or studies, on n-3 intake and fat loss in lean subjects might be too short in duration to show a clinically significant effect. The n-3 supplements simply didn't "kick in", if you will, until the passing of several weeks. Furthermore, any greater weight loss, as a percentage of body weight, is always harder to spot in lean subjects in comparison to obese subjects, unless you have a large sample size (Brilla, et al., used only 8 subjects in the fish oil + exercise group). 

In conclusion, there simply aren't any good trials on n-3 supplementation and their role in fat loss yet. Oh sure, there are studies looking at fish intake and so forth, but I'm talking controlled interventions without a bunch of variables (fish protein, outpatient trials with poor control, etc.).


Omega-3 Fatty Acids: What We Know So Far


Furthermore, research is also highly suggestive of a positive effect of n-3 on brain function and several disease processes, ranging from cancer to the metabolic syndrome. Keep in mind that it's impossible to provide hard data on these aspects of human health. You can't put people in a lab, give some of them some fish oil pills, and look at who got Alzheimer's or cancer 8 weeks later.

Instead you look at things like fish consumption in thousands of individuals, sometimes over several years, and compare the disease trends relative to fish or seafood consumption. When you do this, you often see positive associations between n-3 saturation of the phospholipid membranes and greater health.
This positive association seems independent of other factors that affect health; meaning that, in an imaginary scenario where we compare two people with the exact same stats, such as body fat percentage and lifestyle habits, with the exception of n-3 intake or fish intake, the person with a higher n-3 intake will be healthier.

A striking example of this, is the fact that fat Eskimos have significantly lower rates of Diabetes Type 2 compared to equally fat Americans (3.3% vs 7.7%). Not a completely valid comparison, due to the inherent problems of dietary epidemiology, but it illustrates the point I'm trying to make, as these health trends are seen across the board in many different populations.

It's safe to say that n-3 is good for us. The Western diet contains too little omega-3 relative to omega-6 fats for optimal health, and our low n-3 intake might certainly be an independent factor influencing the disease trend amongst Westerners compared to other populations with high n-3 intake (e.g. Eskimos, etc.).

Now finally, what about those new studies on n-3 and muscle protein synthesis? 

I saw that Michael Rennie, a hot shot in protein research, was among the authors so I very was excited to read it. The results were very interesting.
This study was carried out on older adults, aged 65 and above. The elderly population is a high-priority group in protein research and there are probably more studies looking at optimizing protein intake for the elderly than there are studies looking at how to make young guys or athletes ripped and swole. How come?

As we age, we encounter a phenomenon called "anabolic resistance". This is a catch-all term for an overall crappier response to weight training and dietary protein.

A fancier sounding word for anabolic resistance is "sarcopenia". But this is just a made-up word that researchers use to get funding for studies. Sarcopenia sounds better than anabolic resistance, I guess.

In terms of dietary protein, "leucine resistance" describes the phenomenon in regards to how the response to feeding is altered. The amino acid leucine is the primary amino acid responsible for triggering muscle protein synthesis. Elderly folks need higher concentrations of leucine to get the same response as young folks. Various feeding strategies that overcomes this problem include:

1. "Protein pulse feeding", which means eating infrequent meals higher in protein and leucine (in contrast to frequent meals). There is a threshold level of leucine needed to consume in each sitting to maximize muscle protein synthesis and this threshold is raised with aging. Larger meals cause leucine to hit that threshold, which triggers muscle protein synthesis. Leucine resistance is similar to insulin resistance in the sense that more leucine and insulin is required to trigger the same response.

2. Supplementing with whey protein or amino acids (EAA, BCAA or leucine). There are many practical advantages of this strategy, i.e. the hassle-free process of drinking whey protein or taking free-form amino acids.

3. Simply eating enough high-quality protein in each meal. This might seem like a no-brainer for us, but with aging comes a loss of appetite. When was the last time you saw a 70-year chew down a big steak? Achieving a high leucine concentration in the blood is mainly about eating enough protein. In the case of elderly folks, faster protein sources - such as well-chewed meat or whey protein - is preferable. This is first and foremost due to absorption kinetics; i.e. leucine concentrations will rise fast and the threshold will be easily reached with 20-30 g protein.  

So that's that. As a side-note, I detest faux diet gurus who cite studies on protein supplements for the elderly to sell leucine supplements. It makes zero sense for a young guy on a high-protein diet.

(Context is crucial. The supplement industry flourishes because no one cares to read or question their claims. Here's some advice: take a second or two to actually read and reflect on studies that are cited in order to validate a claim ("Leucine increases muscle protein synthesis by 537%!"). If it sounds too good to be true, it is too good to be true.)

Anyway, so this one study I mentioned earlier showed that 8 weeks of n-3 supplementation lead to a significant increase in muscle protein synthesis in response to feeding. The same experiment was repeated with younger subjects and the results were released a few weeks ago. 

Below, I'll do a detailed review of the newly released second study and talk a about the results.

Omega-3 Fatty Acids and Muscle Protein Synthesis


Here's the study I'm going to talk about now:

Note that "young" in a clinical context is not necessarily young as we tend to think of it. The average participant was almost 40-years old. Clinical standards are somewhat different than those used in everyday speech. For example, "lean" usually means 15-20% body fat, "high protein" tends to be used liberally and for almost any diet regimen with >0.8 g protein/kg (FDA standard), and a 30 g protein meal is considered a "high protein meal", and so forth. 

After obtaining baseline values for anabolic signalling pathways (mTOR-p70s6k) and muscle protein synthesis, the participants were given 4 g n-3/day for 8 weeks.

...the effect was probably mediated via one or more alternative pathway(s), which have yet to be determined (but may include e.g., Rheb or vps34).

Rheb is a cell membrane protein and vps34 can roughly be described as a nutrient sensor. Both of these are involved in activation of the anabolic cascade that follows amino acid sensing outside the cell.

Furthermore, weight training also activates these anabolic triggers, which has the effect of increasing MPS more than normally, i.e. the cell takes up more amino acids from the same amino acid dose if Rheb/vps34 expression is increased from weight training. The speculation above revolves around whether n-3 supplementation has the potential to alter Rheb/vps34 expression.

Considering the observed changes in skeletal muscle phospholipid composition, it is also possible that LCn-3PUFA supplementation modulated key substrates along the anabolic signalling cascades by affecting membrane lipid composition and/or fluidity.

Another potential mechanism, albeit very poorly and unprofessionally formulated in my opinion. Amino acids do not simply diffuse and move through the cell membrane; "fluidity" should not affect uptake of amino acids in that sense.

However, n-3 enrichment of the membrane has the potential of affecting nutrient transport via modulation of the integral plasma proteins. Go back and read about the avocado if you don't follow. Look at the white pins. Some of them are amino acid transporters.

I believe one potential mechanism for the increase in MPS post-n-3-supplementation is altered amino acid transporter expression. Amino acid transporters  are in contact with the cell membrane, and can translocate to the membrane surface in response to amino acids in the blood plasma. It's possible that an n-3 enriched membrane sensitizes, increases, or somehow alters transporters such as System-L, which is primarily responsible for uptake of leucine.

(Interesting side-note: paradoxically, transporters also increase during amino acid deprivation; i.e. starvation - presumably for transporting amino acids out of the cell interior, or to allow rapid resynthesis of muscle protein once amino acids become available again.).

A second hypothesis of mine, albeit somewhat remote and vague, is that increased blood flow supplementation might have something to with it. I do not have enough insight into hemodynamics to provide an informed opinion, but perhaps n-3 supplementation affected blood flow in such a way that hyperaminoacidemia occurred faster.

However, this would only alter the temporal pattern of MPS, and not the absolute rate. That might be a desirable effect for the elderly population, but not necessarily great for younger folks.

It is unlikely that the beneficial effect of LCn-3PUFA on muscle protein synthesis was related to their antiinflammatory properties because our subjects were young and healthy and we did not detect any treatment-induced changes in inflammatory cytokine concentrations in plasma – most likely because the concentrations were very low to begin with.

One hypothesis was that MPS could be negatively affected by inflammation. Since these subjects were healthy, there was no meaningful relationship between MPS and these markers. However, low-grade inflammation is often present in metabolically challenged individuals and there are studies which suggest that MPS is impaired in these cases.

Lowering inflammation, i.e. via n-3 supplementation, might be beneficial for restoring MPS in inflammatory states, such as obesity or illness. I don't think any human studies have confirmed this as far as obesity goes, e.g. lowered inflammation with n-3 and seen increases in MPS, but it's a theory.

Lowered inflammation with n-3-enriched tube feeding was posed as a possible mechanism for greater muscle retention in an earlier study on n-3 and cancer (a highly inflammatory disease state.).

...we infused amino acids and insulin at rates close to those used to achieve the half-maximal amino acid induced increase in muscle protein synthesis...

This is in reference to the choice of the amino acid dose to achieve a 50% increase in MPS, which is typical of that dose, based the response seen in other studies. This was done to avoid the "ceiling effect".

The "ceiling effect" is also referred to as the "muscle full" phenomenon and denotes the point where MPS falls back to basal values. In the Average Joe, this ceiling is reached in the third hour in response to an infusion rate of 10 g amino acids/hour, which amounts to about 2 g leucine total. After that, MPS becomes refractionary. A logical response. Had MPS not fallen back to basal values, we'd all look like Ronnie Coleman times ten.

Simply put, there is a limit to how much amino acids the muscles require for maintaining normal function, and they will not take up more aminos than what is required for muscle homeostasis.

We made our measurements of muscle protein synthesis during a 3-h infusion of insulin, amino acids and glucose because the rate of muscle protein synthesis rises quickly (within <30 min) in response to increased amino acid availability but then returns to basal values after ~2.5-3.0 h.

Important: This is a crucial aspect of this study that might make the results less than fully applicable. In fact, I think the researchers have made a critical error here.

It seems that they choose the amino acid infusion rate (105 mg/kg/hour) based on the assumption that MPS becomes refractionary in the third hour, regardless of the infusion rate, which is not the case at all. Bohe et al., showed that the refractionary response occurred in response to an infusion rate of 162 mg/kg/hour. Meaning that they had no grounds for limiting themselves to a 3-h infusion.

But why is this important then? Because the short time-period vastly limits the conclusion that can ultimately be drawn from the results. For example, recall that MPS increased by 68% in response to amino acids pre-n-3 supplementation. After n-3-supplementation, the same dose caused a 98% increase.

However, nothing can be said about the temporal pattern during these 3 hours. What if MPS would have become refractionary in the 4th hour after the second experiment, whilst remaining elevated after the first experiment?

We can only say that the absolute increase in MPS was greater within the narrow time-period of the study protocol. However, recall what I said about the "muscle full" scenario. It might be that n-3-supplementation simply increases the rapidity of amino acid uptake for any given dose; e.g. by altering amino acid transporter expression, which would then result in greater MPS within a shorter time-period.

However, once "muscle full" is achieved (after the absorption of ~2 g leucine), it should theoretically become refractionary. In contrast, sans n-3-supplementation the absorption would be slower, resulting in lower MPS that becomes refractionary at a later stage.

The key question here, which in my opinion remains unanswered, is whether n-3-supplementation has the potential of increasing the absolute capacity for MPS in response to a fixed amount of amino acids; similar to what weight training does. There is some indication that this might be the case:

There was a trend for an increase in the RNA-to-DNA ratio, the cell capacity for protein synthesis, but the difference did not reach statistical significance (P = 0.13).

Important: If this is the case, n-3-intake would be one of the most important dietary components for body composition. I am not much for hyperbole, but this is a very exciting study - even if it's limited by its short infusion protocol.

Therefore, we assume that the increase in the anabolic response after LCn-3PUFA supplementation was due to an increase in the magnitude of the anabolic response. However, we cannot rule out the possibility that the effect was due to an increase in the duration of the anabolic effect of nutritional stimuli.

Yes, as I noted before, they cannot say much about the temporal pattern. I'm disappointed in Rennie for his lack of critical reflection around the short infusion protocol, which was chosen based on erroneous assumptions in regards to the refractionary state. Then again, even the best make mistakes.

This study could have been vastly improved if they had chosen the infusion rate (162 mg/kg/hr) required to reach a "muscle full" scenario within three hours and used a 4-hr infusion protocol. They would then compare MPS pre- vs post-n-3-supplementation. Had they then seen that MPS was higher post-n-3-supplementation, their conclusion...

In summary, we have shown that LCn-3PUFA supplementation in healthy 25 - 45 y old individuals increases mTOR signalling and the anabolic response of muscle protein synthesis to hyperinsulinemia-hyperaminoacidemia, which resulted in increased muscle cell size (protein-to- DNA ratio) and protein concentration.

...would have been a lot more valid.

The specific mechanism(s) by which LCn-3PUFA act on the muscle protein synthesis process remain mostly unknown.

And the mechanism by which omega-3 fatty acids affects muscle protein synthesis remains shrouded in mystery.





Here's the reason I don't eat much fish. I have nothing against fish, but there is no protein source I would rather choose over roast beef. Perhaps one day we might also uncover the anabolic potential of roast beef. But until then, I'll make sure to supplement my fat intake with EPA and DHA. I suggest you do so too, if you don't eat fatty fish on a regular basis.



Final Thoughts and Recommendations


Even though the conclusions that can be drawn based on this study are somewhat limited due to the infusion protocol, I find the results promising enough to make changes to my own diet. Consequently, I will personally increase my daily n-3 intake to achieve a similar EPA/DHA saturation of the plasma membranes. I will also make this a mandatory part of the diet templates for my clients.

This means that I recommend an intake of 2 g EPA and 1.5 g DHA per day, and it means that you should pay closer attention to the EPA/DHA amounts you get from your fish oil supplement.

As I mentioned earlier, a 1-gram fish oil capsule does not yield high amounts of EPA and DHA, as most of it is filler. The 3 grams of fish oil I have been taking up until now yields a trivial amount of 0.18 EPA/0.12 DHA per capsule. I'd need about 10 of those capsules to reach the amounts required.

Which fish oil supplements contain satisfactory amounts of EPA/DHA? I don't feel like chewing on 10 of those capsules, so I will explore other alternatives. I've looked around for a bit and have found these high-EPA/DHA-brands:

NutraSea Original Liquid.

Cost of daily dose required (3 tsp/15 ml liquid) to reach 2 g EPA/1.5 g DHA:  $0.99. (500 mL bottle.)

Ultra Omega-3

Cost of daily dose required (4 softgels) to reach 2 g EPA/1.5 g DHA: $0.44. (180 g softgels.)

Swedish readers: "Omega-3 Forte" from Pharbio seems to be the best choice. You will need 6 softgels to meet the EPA/DHA-requirements. Daily cost: 9.4 SEK. You can order Omega-3 Forte from Svenskt Kosttillskott

The aforementioned brands are good and affordable alternatives for meeting the daily EPA/DHA requirements. Softgels are always cheaper, but some people can't stand fish oil capsules, in which case a liquid solution might be a more attractive alternative. So that's that.

If you eat fatty fish on a regular basis, a stringent EPA/DHA-protocol might not be required. You can check the EPA and DHA content of fish here. Example: your average store-bought salmon yields 0.7 g EPA and 1.4 g DHA per 100 g. Do the calculations based on your average weekly intake. Keep in mind that the amounts can vary depending on the breeding of the fish, e.g. farm-raised vs wild salmon.

Read more about supplements you might actually find useful in this article.

---

Where have you been? 

Some readers might wonder why there haven't been any updates for more than a month. I blame this partly on my new laptop crashing. I bought it for myself as a Christmas present, but it came with an unfortunate defect in the HDD which caused my laptop to crash.

The process of sending it back and forth to repairs took about three weeks, during which I had to used my old laptop (which is ridiculously slow). When I write, I always work with several tabs open, since I need to do frequent fact-checking for the studies I cite, and so forth. I couldn't stand doing that on my sluggish old laptop. My work and motivation suffered.

Once I received my new laptop in return again, I had fallen out of the groove of writing and just got lazy and complacent. But I'm back on track now, it seems. Hopefully, you won't have to wait another 5 weeks for the next update.

I hope you found today's article interesting. I apologize for the ranting, the technical detail, and the length of the study review. I think the results are truly interesting, and perhaps revolutionary as far as supplements goes. I was eager to give you the full scoop without dumbing it down too much.

If you enjoyed the article, or any other article on my site for that matter, perhaps you might consider a donation in show of support of my writings and the time I put into them.

That's all for today, folks.