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  • 3 months later...

What I have not seen is a direct comparison of the degree of autophagy for time restricted feeding of 8/16 vs 20/4. ... Ditto for prolonged fasting: do 5 day water fast offer anything a consisten 7/16 intermittent fasting does not?

There's next to no data on the human time-course: you'd have to either have transgenic humans expressing fluorescent tags upon expression of key autophagy-induced genes, or have people willing to undergo repeat biopsies over the course of your time period of interest (and be able to take account of the artifactual effects of these injuries). I was able to find one study, but it doesn't really answer your question:


Fasting Increases Human Skeletal Muscle Net Phenylalanine Release and This Is Associated with Decreased mTOR Signaling

Eight healthy male volunteers were studied ... on 2 occasions separated by >1 month: 1) after an overnight fast of 10 hours (control), and 2) after 72 hours of fasting, during which subjects were allowed to drink tap water and to perform normal ambulatory activities, excluding physical exercise. At 0800 h (t = 0) the subjects were admitted to a quiet, thermo-neutral room on both examination days. ... Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.



mTOR phosphorylation was decreased by ∼50% following [72 h] fasting [compared to the control 10 h overnight fast], together with reduced downstream phosphorylation of [mTOR targets] 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic.


However, they just have those 2 time points, and only in muscle: we don't know what the eg. 24 and 48 h induction levels looked like.


There's more data on mice, but even there not with the kind of granularity you might be hoping for (and with the understanding that the effects of extended fasting are likely different in mice due to their lower energy reserves, faster metabolic rate, etc). Humans on regular CR exhibit elevated autophagy in muscle after a normal overnight fast, whereas marathoners and Western dieters do not; on the other hand, an acute bout of "aerobic exercise activates autophagic signaling through ULK1 in human skeletal muscle, independently of nutrient background".


Importantly,  autophagy is engaged to varying degrees in varying tissues even over the course of a normal day with an 8 h overnight fast. Here's what it looks like in mouse liver (remember, or know ye, that mice are active in the dark and sleep in the day, so invert the pattern you see here):



Figure 1. Rhythmic induction of autophagy in the liver. "Temporal orchestration of circadian autophagy rhythm by C/EBPβ."


Mice tend to either max out autophagy at 24 hours of fasting, or slightly increase it, or return to baseline at 48:



Figure 7. Quantitative analysis of the formation of GFP-LC3 dots during starvation.  In Vivo Analysis of Autophagy in Response to Nutrient Starvation Using Transgenic Mice Expressing a Fluorescent Autophagosome Marker


.. with the caveat that we don't have data for eg. 12 or 36 hours. Here's a similar study in mouse cortical neurons:


Autophagosomes are detectable in cortical neurons of normal-fed mice, and their abundance and characteristics are markedly altered by short-term food restriction.

... Autophagosomes were readily detectable in cortical neurons of normal-fed mice  ... These vesicles were few in number and small in size (Fig. 2B). Food restriction caused a marked increase in both the number and the size of neuronal autophagosomes in the cell body of cortical neurons; these changes were present after 24 hours of food restriction (Fig. 2A, middle row), and were even more dramatic at 48 hours (Fig. 2A and bottom row). As was observed in the liver (Fig. 1), when compared to autophagosomes in normal-fed mice, those present in the cortical neurons of mice that had been food-restricted for 48 hours showed reduced circularity, with concomitantly increased feret and perimeter (Fig. 2B). Changes in GFP-LC3 signal also were observed in the neuronal cell processes ...

(see their Figure 2 and Figure 3).


As you can see, there is baseline autophagy in cortical neurons of fed mice, which increases with fasting; most of the induction is present at 24 h, with a more modest further enhancement at 48.


This is all macroautophagy, by the way: the circadian rhythms of micro- , chaperone-mediated, and mitophagy doubtless all follow a different pattern (see wikipedia if this is gibberish).


A related question is - Michael has presented arguments that CR has all the same benefits of periodic fasting ( and is indeed the mechanism) and induces a gradual cumulative autophagy vs intense rapid autophagy achieved by periodic fasting.


.... which is then even more profoundly turned off upon refeeding. And per unpublished work by Ana-Maria Cuervo, "regular" 40% daily CR is very effective in boosting CMA, whereas alternate-day fasting has nearly no effect (unpublished, but presented at UABBA 2008).



I am not aware of any data showing their relative cumulative effects - This begs the question whether an intense autophagy has any unique benefits over prolonged continuous autophagy ( besides refeeding hematologic/stem cell regeneration). I wonder are there any insights or other data out there I am aware of contrasting these?

If intense autophagy has any unique benefits over prolonged continuous autophagy, it would make the mice live longer — right?

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  • 2 weeks later...
On 5/17/2017 at 6:10 PM, Mechanism said:

I can't seem to link to it but "Effects of intermittent fasting on body composition and clinical health markers in humans" provides a great review of the limited data available and describes a rapid acceleration in alterations of lipid and glucose metabolism (as glycogen is depleted) ... primarily between around 12-18 hours of fasting, with more modest additional benefit in the 18-24 hour fasting period. This change in fuel source seems to be part of the trigger for autophagy.

The paper is uncharacteristically available free from the publisher. It does look good from a quick glance. It's unfortunate that it wasn't published a year or 2 later, as a signifcant am't (much of it by Tinsley himself) of human research on 18:6 and 20:4 has been published since.

We may get a better idea on this soon. I was recently alerted to this paper (you'll have to copy-paste the link: for some reason we're not allowed to link Taylor & Francis' website on the Forums):


... which reports a "Transient and robust amino acid release (amino acid surge) in liver, blood and skeletal muscles" in fasted mice at 24 h, corresponding to a peak in other markers of autophagy in the liver. If this is confirmed, researchers could begin using such a surge as a surrogate marker for a surge in autophagy, and we'd be able to track the time-course of macroautophagy, at least, during fasting without needing genetically-modified humans or a lot of invasive unpleasantness.


On 5/17/2017 at 6:10 PM, Mechanism said:

On the other hand, Valter Longo has been quoted in a follow-up to a podcast interview that for autophagy "3 days is optimal for mice. For humans 4-5 minimum, depending on what you are trying to achieve”" (source:http://joshmitteldorf.scienceblog.com/2014/06/12/the-three-day-fast-day-four/). I am not sure what he means by "trying to achieve."

Me neither — and in particular, I'm not sure whether they're relevant to otherwise-healthy aging people, nor what his evidence for it is.

On 5/17/2017 at 6:10 PM, Mechanism said:

The citations you provide above are interesting though I'm not clear on whether a series of shorter 1-day fasts can add up to the benefits of less frequent longer fasts or whether there are qualitative metabolic differences (advantages) of a longer fast that cannot be well replicated by a series of shorter fasts.

Me, either. There seems to be a widespread assumption that longer fasts lead to greater qualitative/categorical benefits, with no clear evidence base for it. More importantly, they seem to neglect the obvious possible downside of extended fasting, in which the body is catabolizing its muscles and not engaged in anabolic activity in the bones for longer and longer periods, or prolonged impairment of neurogenesis in the brain. (To be clear, I have zero evidence for the latter — I just suspect that a sufficiently prolonged lack of anabolic signaling would impair cell division globally, including brain and other tissue stem cell division and mobilization, to one's long-term detriment).

On 5/17/2017 at 6:10 PM, Mechanism said:

I think I know your answer: while qualitative difference may exist there has been no direct comparison between regular daily time restricted feeding and less frequent prolonged fasts. Therefore the only safe bet is CR which is better established, and regular 24 hour fasts may very well work just as well or better than less frequent prolonged fast but nobody knows and (as you have articulated before) there is no good evidence that either of these work above and beyond the degree of CR achieved so it is may be a moot point... do Inhave that right?

I agree entirely with your first sentence. I'm not sure whether or not to agree with "Therefore the only safe bet is CR," because it depends on what contrast you're drawing. If you're contrasting CR vs. no reduction on net energy intake with meal patterning and the goal is the retardation of aging, then absolutely, yes. If you're contrasting regular daily CR with isocaloric EOD/IF, then I'd say both are equally safe bets: per the rodent data, it's a wash. And there is some evidence for a metabolic benefit to time-restricted feeding, even with no energy restriction, in people with substantial metabolic syndrome — particularly if combined with proper circadian timing (tho' don't ask me to say what "proper circadian timing" is exactly: I suspect it means "first thing in the morning," but the studies are inconsistent). But, none of this as against aging and lifespan in normal, healthy people.

On 5/17/2017 at 6:10 PM, Mechanism said:

I smiled with your last comment "If intense autophagy has any unique benefits over prolonged continuous it would make the mice live longer — right?".


My primary objective right now is to live relatively free from chronic disease and other impairments of quality of life ( QoL) rather than extend my life beyond that seen in very healthy populations such as in the Blue Zones. So I would settle for compression of morbidity, especially if it materially lessens my risk for cancer

This is the old healthspan-lifespan false dichotomy. People or mice who live relatively free from chronic disease and other impairments of quality of life with materially lessened risk for cancer don't just drop dead: if you're healthy, you keep living, unless you're hit by a car or murdered in your bed, whereas if you're plagued by chronic disease and cancer, your risk of death increases. This is all the more so in contemporary industrialized countries, where your risk of premature mortality from age-related causes is low if you have access to healthcare and do the basic minimum on the lifestyle front.

On 5/17/2017 at 6:10 PM, Mechanism said:

hence my focus on lower protein and amino acid nutritional geometry, maintaining low IGF-1, and mechanisms of autophagy. At this juncture, forgoing perhaps several extra years or so of extra life relative to healthy populations such as the 7th day Adventists is a small price to pay on my utility curve, given that I do not ascribe to the "escape velocity" hypothesis, at least for my lifetime.

The Adventist data has been substantially exaggerated; some time I must properly reply to the thread on this. IAC, supposing (again) that you were one of these super-healthy 80-year-olds who was mysteriously just about to drop dead of nothing in particular, I guarantee you'll want that extra year of life when your biological intrinsic lifespan runs out.

On 5/17/2017 at 6:10 PM, Mechanism said:

Also for rationale, having greater caloric intake relative to a formal CR practice may preserve immune function better than full CR ( noted by Aubrey on the Okinawans on his interview with Rhonda Patrick) and/or enhance hardiness/robustness against frailty and associated morbidity and QoL impairment from sarcopenia, etc.

As has been extensively discussed, CR is protective against sarcopenia: yes, CR animals have less muscle than AL animals while young, but the quality of that muscle is better-preserved than either AL sedentary or even AL exercised animals, and the quantity holds steady against age-related decline.


[Old Wikipedia Entry]

Calorie restriction preserves muscle tissue in nonhuman primates[71][72] and rodents.[73][74] Mechanisms include reduced muscle cell apoptosis and inflammation;[73] protection against[74] or adaptation to[71] age-related mitochondrial abnormalities; and preserved muscle stem cell function.[75] Muscle tissue grows when stimulated, so it has been suggested that the calorie-restricted test animals exercised more than their companions on higher calories, perhaps because animals enter a foraging state during calorie restriction. However, studies show that overall activity levels are no higher in CR than AL animals in youth,[76] while CR animals are more active at middle age and beyond due to a protective effect against the decline in activity observed in middle-aged and older animals.[77]

Work in the heart also finds that CR inhibits fibrosis, and they found reduced skeletal muscle fibrosis in "CR" primates at WNPRC. Etc.

On 5/17/2017 at 6:10 PM, Mechanism said:

Conceptually, with limited long-term data in humans, a weaker form of CR with restricted eating windows to enhance autophagy may reduce the risk for cancer while also providing some insurance of keeping a bit of distance from the statistical tails by maintaining a body habitus more closely resembling that in the range of traditional Okinawa and 7th Day adventists (groups with borderline underweight BMI in the 17's for the former, to lower to mid-range of "ideal" BMI for the latter respectively) for the long-term while also improving quality of life in the short term.

But you're substituting even weaker evidence for the uncertain case for CR. And, you're mixing the CRed Okinawans with the lean AL Adventists, which is really apples and oranges, and focusing on population BMI as an individual target, which is misguided. People who are "naturally skinny" are not ipso facto on CR.

On 5/17/2017 at 6:10 PM, Mechanism said:

I'm not sure his BMI but Mark Mattson appears to maintain a similar balance as I do right now - a lower BMI with accompanying borderline/modest caloric restriction (though In my case not restricted in calories but rather slim from high BMR and exercise / energy balance), while he practices TRF with daily meals. What's your take on his approach, which closely resembles my own AFAIK?

You'll not be surprised to learn that I think the key question is the degree to which he's (and you're) de facto energy restricted ? , followed only secondarily by his animal protein intake.


Profile: Mark Mattson

He limits himself to 2,000 to 2,200 calories a day and runs four miles every day. Breakfast is often nonexistent, lunch is light, and dinner—which is different from his family's—is typically a bowl of steamed broccoli, carrots and cabbage with some salmon or yogurt. Mattson says he indulges himself on special occasions and, often before bed, gulps down a bowl of oatmeal and raisins.



How 5 Longevity Researchers Stave Off Aging

By Lindsay Lyon

Mark Mattson, 52

Chief, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health

What he does: Limits calories to around 2,000 per day. Always skips breakfast, and lunch on Mondays, Wednesdays, and Fridays. Every evening he grazes on fruit, veggies—and a bowl of oatmeal—in addition to dinner.

Why: Research by Mattson and others suggests that restricting calories (and occasionally fasting) can stimulate "adaptive stress response mechanisms" in the body, which may boost its resistance to injury and disease. Doesn't he get hungry? "I think it's actually good to be hungry," says Mattson, who is 5 feet 9 inches tall and weighs less than 130 pounds [bMI <19.2].


On 5/17/2017 at 6:10 PM, Mechanism said:

Thanks for pullling those references - one of the most interesting items you brought up above was the work of Ana-Maria Cuervo - I was not aware of her unpublished research. As a practitioner with a remote background in research I always ask - why was the work not published in a peer reviewed journal?

The results if to believed ( it still would be only one study, though a potentially valuable contribution) is significant in scope & implications if the methodology is reliable. Do you have any insight into this - perhaps limitations in the methodology undermined the reliability of the results? The devil is in the details too, length of intervention, reliability of the data, etc.

No, the study was solid, per Dr. Cuervo. I don't remember the details, but the gist of it was that the graduate student who was most directly involved in the work abruptly took a position at another institution and was busy with something else, and between her and the people and data back at Dr. Cuervo's lab they were never able to get themselves together to properly write it up.

It's "only one study," yes — but it's the only study, and Dr. Cuervo is one of the world's leading experts on autophagy generally, and probably the leading person doing work on aging and CMA.

On 5/31/2017 at 6:00 AM, Mechanism said:

Al found a thread supporting some unique benefits of IF - even over CR - quoted below ( context of thread above looking at autophagy captured by IF ) - nice find Al! Would be interested in any perspectives on these findings in the setting of looking to optimize autophagy.

Comparison of Intermittent Fasting Versus Caloric Restriction in Obese Subjects: A Two Year Follow-Up.

Aksungar FB, Sarıkaya M, Coskun A, Serteser M, Unsal I.

J Nutr Health Aging. 2017;21(6):681-685. doi: 10.1007/s12603-016-0786-y.

PMID: 28537332

As you note separately, this has limited applicability as this wasn't CR, but weight loss in the obese: as you note, they had already been restricting energy for nearly a year when they began the Ramadan IF regimen, and were apparently still losing weight throughout it. Note that, as one might expect when obese people lose weight, IGF-1 levels were actually rising during the initial period of energy restriction.

On 5/31/2017 at 6:35 AM, Sibiriak said:

Cf. Comparison of intermittent fasting versus caloric restriction in obese subjects: A two year follow-up

The Journal of Nutrition Health and Aging August 2016

Full text available:


That's the same paper: the one dated for August in ResearchGate is the early-release electronic-only publication, whereas the one dated for this year is the final version formatted for print and released along with it. You'll note as one clue the unlikely pagination of the epub version.

A better study — because it had more people, a conventionally-restricted control group, a lower dropout rate, better-described methods, and started with people who were (sort of...) metabolically healthy if still obese — was this Longo-style modified fasting study by long-time fasting diet researcher Krista Varady:


Effect of Alternate-Day Fasting on Weight Loss, Weight Maintenance, and Cardioprotection Among Metabolically Healthy Obese Adults: A Randomized Clinical Trial.

Trepanowski JF1, Kroeger CM2, Barnosky A1, Klempel MC1, Bhutani S1, Hoddy KK1, Gabel K1, Freels S3, Rigdon J4, Rood J5, Ravussin E5, Varady KA1.


Importance: Alternate-day fasting has become increasingly popular, yet, to date, no long-term randomized clinical trials have evaluated its efficacy.


Objective: To compare the effects of alternate-day fasting vs daily calorie restriction on weight loss, weight maintenance, and risk indicators for cardiovascular disease.

Design, Setting, and Participants: A single-center randomized clinical trial of obese adults... 100 participants (86 women and 14 men; mean [sD] age, 44 [11] years),... mean body mass index, 34)  ... randomized to 1 of 3 groups for 1 year: alternate-day fasting (25% of energy needs on fast days; 125% of energy needs on alternating "feast days"), calorie restriction (75% of energy needs every day), or a no-intervention control. The trial involved a 6-month weight-loss phase followed by a 6-month weight-maintenance phase. ...

Results: ... the dropout rate was highest in the alternate-day fasting group (13 of 34 [38%]), vs the daily calorie restriction group (10 of 35 [29%]) and control group (8 of 31 [26%]). Mean weight loss was similar for participants in the alternate-day fasting group and those in the daily calorie restriction group at month 6 ...and month 12 ... Participants in the alternate-day fasting group ate more than prescribed on fast days [consuming ≈1100 Cal, vs. a prescribed 500 Cal in the first 6 mo and 750 Cal thereafter — Fig. 2A]  and less than prescribed on feast days [≈1600 Cal, vs a prescribed 2100 Cal in the first 6 mo and 2600 Cal thereafter] while those in the daily calorie restriction group generally met their prescribed energy goals.

There were no significant differences between the intervention groups in blood pressure, heart rate, triglycerides, fasting glucose, fasting insulin, insulin resistance, C-reactive protein, or homocysteine concentrations at month 6 or 12. Mean high-density lipoprotein cholesterol levels at month 6 significantly increased among the participants in the alternate-day fasting group (6.2 mg/dL [95% CI, 0.1-12.4 mg/dL]), but not at month 12 (1.0 mg/dL [95% CI, -5.9 to 7.8 mg/dL]), relative to those in the daily calorie restriction group. Mean low-density lipoprotein cholesterol levels were significantly elevated by month 12 among the participants in the alternate-day fasting group (11.5 mg/dL [95% CI, 1.9-21.1 mg/dL]) compared with those in the daily calorie restriction group.

PMID: 28459931

Ie, once again, in this setting: all the benefits are accounted for by net energy intake (and, in this setting, presumably loss of adipose tissue, particularly visceral adipose) — and in this case, it seems to have actually resulted in worse LDL levels by trial's end.

However, while this was a better test than previous studies of the real-world effects of prescribing modified fasting vs. conventional energy restriction for obesity, it's far from an ideal test of the actual metabolic effects of such a diet for people who actually follow it, since (as they note) after the initial run-in 3 months during which the food was provided for subjects, people prescribed the modified fasting diet tended to drift toward something closer to a conventional energy-restricted diet; a more rigorous long-term study would be more informative for the hardy or better-predisposed few who adhere consistently to the program.

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Michael R:  People who are "naturally skinny" are not ipso facto on CR.



Do you have a definitive definition of CR  in humans?      A percentage reduction from "AL" intake would seem problematic since, among other things, it doesn't take into consideration energy expenditure (exercise etc.)(Dean P.'s "net calories" issue),  nor the fact that pre-CR   "AL" intake  in humans can vary for all sorts of psychological and cultural reasons, apart from physiological ones.


I know this has been discussed elsewhere, but perhaps you could just state (or link to)  the simple definition you employ.

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Mechanism: ... for CR definitions I'm going to let Michael answer this one but FYI I found his thread at http://www.longecity...quotidian-diet/which points to http://www.longecity.org/forum/topic/45958-calorie-restriction-targets/#entry450451 most helpful. 


Thanks for that link!!   I was looking for a clear definition,  i.e. "a person is CR'd  iff..."


What I found there:

  • A person is CR'd if he/she  restricts caloric intake while maintaining adequate nutrition so that he/she  weighs at  least 15% less than his/her biological setpoint weight.
  • Biological setpoint  weight  is defined as  the weight to which one tended to remain stably when in  one's early twenties, and that was within the healthy BMI range.


(Forms of protein/amino acid restriction and various other prescriptions for an optimal diet are additional interventions outside the scope of CR per se.)


I think most interesting is the question and controversy as to what degree it is a matter of caloric restriction versus a net energy intake versus expenditure, something Dean has promoted and debated with Michael in the past


Yes, I mentioned Dean's net caloric intake issue in my question.    The above definition  doesn't directly address it,  unless one reads into it  that the movement to the target  15+% below biological set point weight should be done only via caloric restriction,  not an increase in energy expenditure (beyond some defined moderate- healthy-exercise-produced amount?) or a combination thereof.   That interpretation would raise a number of questions, of course.


Another issue would be the vagueness/variability of the biological setpoint weight value.


Sorry if I'm butting into your excellent discussion!  Ignore at will.

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Mechanism: ... for CR definitions I'm going to let Michael answer this one but FYI I found his thread at http://www.longecity...quotidian-diet/which points to http://www.longecity.org/forum/topic/45958-calorie-restriction-targets/#entry450451 most helpful. 


Thanks for that link!!   I was looking for a clear definition,  i.e. "a person is CR'd  iff..."


You're not going to get one ;)xyz. Go be a lab mouse ;)xyz. Sorry: that's just the best we can do.


Happily, you seem to have quite correctly understood the issue. I've also just pinned a FAQ post: How Many Calories Should I Eat? What's My Goal Weight? What's My Setpoint? What "%CR" Am I?. So, no one will ever ask this question ever again :) .

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Thanks Michael.   To clarify,  I was interested in a definition of CR in humans  in relation to CR science and theory more than in relation to personal CR practice.    I would think that  good CR science would require clear definitions. 

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Thanks Michael.   To clarify,  I was interested in a definition of CR in humans  in relation to CR science and theory more than in relation to personal CR practice.    I would think that  good CR science would require clear definitions. 


There's no "science theory" about it. CR is defined operationally, as laid out in that post. What's the science theory of "is taking rapamycin"? It's someone who swallows this particular pill every day.

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CR is defined operationally


I never said it wasn't.  ("CR Science and Theory" is the title of this forum.)  A good operational definition still demands clarity.   Your new FAQ post is helpful in that regard.  Thanks.



Heh? How is it not helpful? It's quite precise on the operational definition — in rodents. If you're asking about human practice, it's not — but you just said you weren't asking about human practice :wacko: . EDIT: Michael needs to actually read the post to which he is replying. Apologies, Sibirak. Glad you did find it useful. ;)xyz

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