REF-1557 · Granata et al., 2016 · FASEB Journal · Paper · PubMed

Do twice-daily intervals raise mitochondria?

In 10 men, 20 days at 2 a day went with respiration and citrate synthase up about 40-50%; most no longer differed 2 weeks later. No same-volume once-daily arm: dose, not the split.

Main caveat: No control, fixed order, so time, carry-over and dose cannot be separated.
limited, mixed evidenceMeasured on non-athletes and recreational people

Ten healthy men cycled high-intensity intervals through three consecutive phases: normal volume for 4 weeks at 3 sessions a week, high volume for 20 days at 2 sessions a day, then reduced volume for 2 weeks with 5 sessions. Resting muscle was biopsied from the vastus lateralis at baseline and after each phase. After the first phase nothing mitochondrial had changed.

After the high-volume phase, respiration and citrate synthase were about 40-50% above baseline, electron transport subunits about 10-40%, and PGC-1α, NRF1, TFAM, PHF20 and p53 about 65-170%; respiration per unit of mitochondria did not change.

After the reduction, all measures except citrate synthase (about 36% above initial) were no longer significantly different from baseline. Our reading: raising volume through twice-daily sessions went with a large rise in these markers, and dropping it went with most no longer differing from baseline within 2 weeks, in a fixed order and with no control.

What it isolates is dose across phases. No arm reached this volume on one session a day, so it cannot credit the split.

Protocol

Design: fixed sequence, no control group, no randomisation of phase order.
Who: 10 healthy men; age and fitness are not in the abstract.
Training: high-intensity interval cycling at 3 a week for 4 weeks, 2 a day for 20 days, then 5 sessions over 2 weeks. Interval prescription is not in the abstract.
Our arithmetic, assuming no day off in the 20: 12 sessions, then 40, then 5.
Measures: mitochondrial respiration, citrate synthase activity, electron transport subunits, PGC-1α, NRF1, TFAM, PHF20 and p53 protein, from resting biopsies.
Not in the abstract: food, glycogen, gap between the two daily sessions, rest days, performance or VO2max figures, how long after the last session the biopsies came.
Funding and interests: Crossref and Europe PMC list the Judith Jane Mason and Harold Stannett Williams Memorial Foundation. No interests statement could be read.

Key Results

MeasureAfter 4 wk, 3/wkAfter 20 d, 2/dAfter 2 wk, 5 sessions
Any mitochondrial parameterno change
Mitochondrial respirationabout +40-50% vs baselinenot significantly different from baseline
Citrate synthase activityabout +40-50% vs baselineabout +36% vs initial
Electron transport subunit proteinabout +10-40% vs baselinenot significantly different from baseline
PGC-1α, NRF1, TFAM, PHF20, p53 proteinabout +65-170% vs baselinenot significantly different from baseline
Mitochondrial specific respirationunchanged
Our arithmetic: the last phase averages 2.5 sessions a week, close to the 3 a week that had raised nothing in the first phase.
Fixed order. The large rise comes second in the sequence and could include carry-over from the first phase. The fall comes third.
"Not significantly different" is not "back to baseline". In 10 men it may hide a remainder; the title's "reversed" is a stronger word than a non-significant test carries.

Mechanisms

Measured: content and respiration moved together while respiration per unit of mitochondria did not. That suggests, by inference, more mitochondria rather than better ones.
Authors' interpretation: training volume is an important determinant of mitochondrial adaptation, and human muscle reverses rapidly when volume is reduced. With no control and fixed order, this is their reading of a sequence.
Not measured, as far as the abstract shows: performance, acute signalling after a second session of the day, the effect of the gap or glycogen.

Practical Application

Do twice-daily intervals raise mitochondria? In 10 men, 20 days at 2 a day went with respiration and citrate synthase up about 40-50%; most no longer differed 2 weeks later. No same-volume once-daily arm: dose, not the split.
Good for: describing how far markers can move with a very large interval volume in healthy men, and how soon most no longer differed from baseline when the volume dropped.
Good for: a caution that a rise in markers can be short-lived. Not from this paper: the base treats a session as a brief signal in PHYS-18.
Not good for: two sessions a day against one. There was no once-daily arm at matched volume, so the gain cannot be credited to the split.
Not good for: copying the dose. About 40 interval sessions in 20 days (our arithmetic), with no performance reported in the abstract. Nothing here says it was tolerable or useful for an athlete in a season.
Not good for: any taper claim. Markers, not performance, were followed, and 5 sessions in 2 weeks is not a designed taper.
Not good for: women or trained athletes by direct reading. Neither is described.

Limitations

No control, fixed order, so time, carry-over and dose cannot be separated.
Marker study. Nothing on performance in the abstract.
n = 10, men only, training status not stated.
Abstract only. Food, gap, rest days and the interval prescription could not be read.
Volume and double together. Going from 3 a week to 14 a week (our arithmetic) changes frequency, weekly dose and likely glycogen at once, so none is isolated.
How this entry was checked and changed
Why this level

an uncontrolled pre-post earns B (rubric), lowered one step: the outcomes are markers rather than performance, and it was read from the abstract only, with no open copy in PubMed Central or Europe PMC