Short version: Two investigational peptides targeting mitochondrial steroidogenesis were safe but did not meaningfully raise total testosterone at the tested dose. One showed a tentative signal on free testosterone. The null result is likely real, and informative.

Context

If you want the full background on why mitochondrial cholesterol transport matters, how VDAC1 and 14-3-3ε regulate steroidogenesis, and why these peptides looked promising preclinically, I covered all of that in detail here:

This post is about what actually happened when I tested the idea in a controlled human experiment.

The Question This Study Actually Asked

Stripped of theory, the experiment boiled down to this:

If you remove an inhibitory constraint on Leydig-cell steroidogenesis, does testosterone meaningfully increase in a real human under tightly controlled conditions?

Study Design

• Design: Randomized, double-blind, placebo-controlled N-of-1 crossover

• Interventions:

  • ACE-167

  • N163-166 Mod. 6

  • Placebo

• Dose: 30 mcg/day (deliberately conservative, first-in-human)

• Phases:

  • 3 × 14-day treatment periods

  • 7-day washouts

• Primary endpoint: Morning fasting total testosterone (LC-MS/MS)

• Secondary endpoints:

  • Free testosterone (equilibrium dialysis)

  • LH, SHBG, estradiol

  • Daily patient-reported outcomes

• Analysis: Bayesian pooled-replicates model (appropriate for N-of-1 inference)

If you want the full protocol, priors, diagnostics, and sensitivity analyses, those live in the manuscript.

🧪 Study Timeline

The Participant

• Healthy 27-year-old male

• Baseline testosterone: 943 ng/dL (>90th percentile)

• Stable sleep, diet, activity

• No alcohol, no endocrine disease

• On finasteride (stable for >2 years)

This was admittedly not an optimized population for seeing large gains.

Primary Result: Total Testosterone

Neither peptide meaningfully increased total testosterone relative to placebo.

Bayesian treatment effects vs placebo:

• ACE-167: −102 ng/dL

  • P(Δ > 0) = 0.06

• N163-166 Mod. 6: +34 ng/dL

  • P(Δ > 0) = 0.73

The probability of exceeding a clinically meaningful increase (~+25%) was essentially zero for both.

Timing caveat: blood draws were fixed at ~2 hours post-dose, extrapolated from rodent oral-dosing data. If peak effects in humans occur earlier, later, or require longer accumulation, this design would miss them. The null result therefore applies specifically to effects present at the 2-hour window.

🧪 Testosterone Trajectories

Why This Null Result Is Probably Real

Two explanations dominate, and they’re not mutually exclusive.

1. Dose was likely too low

The human dose was ~15× below the maximal effective rodent dose after scaling. This study prioritized safety and falsifiability and didn’t attempt to find the maximal effect.

2. Baseline testosterone was already near ceiling

These peptides remove inhibitory processes; they don’t drive production. If Leydig cells are already operating near physiological limits, there may simply be little headroom.

A Mechanistic Clue: LH Went Up, Testosterone Didn’t

Across all phases, including placebo:

• LH increased substantially

• Testosterone stayed flat

• The testosterone-to-LH ratio fell by ~25–45%

🧪 Testosterone vs LH

This pattern is consistent with limited Leydig cell responsiveness at high baseline androgen levels. More signal, same output.

Because LH is highly pulsatile, sparse sampling limits interpretability; observed differences may reflect timing rather than treatment.

The One Signal Worth Discussing: Free Testosterone

Total testosterone was null.

But under N163-166 Mod. 6, free testosterone showed a directional increase:

• +47% vs baseline

• Bayesian estimate vs placebo:

  • +37.5 pg/mL

  • P(Δ > 0) = 0.97

🧪 Free Testosterone by Phase

Important Caveat

• Only two measurements per phase

• Equilibrium dialysis has meaningful analytical and biological variability

• Expected reference change value ≈ 40%

This could be a real signal, or pure noise. Mechanistically, a selective free-testosterone increase without total-T change is hard to explain for this compound class.

Replication with denser sampling is required.

Subjective Outcomes: Interesting, Limited Interpretability

ACE-167 showed higher scores for sexual function and sleep.

But:

• ACE-167 was Phase 1

• Strong phase-order and expectancy confounding

• Autocorrelated daily measures

• Multiple comparisons give randomness a leg up

These are hypothesis-generating only.

🧪 Sexual Function Timeline

Safety

• No adverse events

• No hematocrit rise

• No BP, liver, or metabolic issues

At 30 mcg/day, both peptides were well tolerated, paving the way for future work

What Comes Next (If Anything)

A serious follow-up would need to:

• Use higher doses (60–150 mcg/day)

• Enroll lower-baseline participants (500–700 ng/dL)

• Exclude finasteride

• Sample hormones much more frequently

• Use adaptive Bayesian stopping rules

Whether that’s worth doing is now a much better-informed question than it was before this study.

Bottom Line

This experiment didn’t validate the peptide hypothesis, but it didn’t kill it either.

And in physiology, bounding a hypothesis is often more valuable than chasing another uncontrolled “positive” result.

See below for the full experimental outline and results. Peptide synthesis and supply was provided by Upgraded Bio (admin@upgraded.bio).

Final Report

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Most people think testosterone just… happens. But under the hood, every molecule of it begins life inside a small chemical factory called a mitochondrion, the same “powerhouse of the cell” you probably remember from biology class. Before testosterone can be made, cholesterol has to be transported into these mitochondria. That step is the gatekeeper of hormone production, and that’s where this story begins.

The Mitochondrial Gatekeeper: VDAC1

VDAC1 (Voltage-Dependent Anion Channel 1) is a molecular doorway that helps cholesterol move across the mitochondrial membrane. When it’s working efficiently, cells produce testosterone smoothly. When it’s held back by another protein called 14-3-3ε, that gate tightens and testosterone output can drop.

In rodent studies, disrupting that handshake between VDAC1 and 14-3-3ε caused a clear increase in testosterone, not from adding hormones, but from letting the body’s own machinery work more efficiently.

Meet the Peptides: ACE-167 and N163-166 Mod. 6

Small, stable peptide molecules have been engineered to mimic the part of VDAC1 that 14-3-3ε binds to. When injected, they act like a “decoy” that removes the brake on the system: unlocking cholesterol flow and boosting testosterone naturally.

Two candidates stood out from animal studies:

• ACE-167 (Arg–D-Val–Thr–Gln): Compact and simple, but fast-acting.

• N163-166 Mod. 6 (Ac–Arg–Ile–Thr–Gln–D-Ser–NH₂): A more durable version designed to resist breakdown in the body.

Both elevated testosterone in rats, by as much as 100%, without bypassing the body’s normal feedback control.

The N-of-1 Human Trial: Science, but Personal

Instead of jumping straight to a large clinical trial, this project starts small, just one participant. Me.

The single-participant (“N-of-1”) design includes three 14-day phases:

1. ACE-167

2. N163-166 Mod. 6

3. Placebo

The order is randomized and double-blinded. I won’t know which I’m receiving during each phase.

Every morning, I inject 30 micrograms subcutaneously right after waking, then have blood drawn two hours later on the first and last days of the phase. Testosterone, free testosterone, LH, estradiol, and SHBG are all measured using LC-MS/MS, the gold-standard assay for hormone analysis.

The Goal: A Meaningful Boost Without Replacement

The central question isn’t just “did testosterone go up?” but whether it rose enough to matter. The minimal clinically important difference (MCID) is set at roughly a 25% increase or +236 ng/dL over baseline; large enough to produce noticeable effects on energy, mood, libido, or performance.

The study also asks how this change happens:

• Does LH decrease while testosterone rises (signaling the testicular machinery is more efficient)?

• Does the testosterone-to-LH ratio improve?

• Does free testosterone rise more than total testosterone?

Those patterns reveal whether the peptides are genuinely improving the body’s own steroidogenesis rather than simply altering downstream hormones.

Lifestyle Locked Down

To reduce noise in the data, everything else is controlled:

• Sleep: consistent wake-up times within 30 minutes

• No alcohol or caffeine before tests

• Stable diet and exercise

• All labs at 8:00 AM, at the same facility each time

This level of standardization ensures any hormonal change can be attributed to the peptides, not day-to-day variability.

The Analysis: Blending Old and New Statistics

Results will be analyzed using both frequentist permutation testing and Bayesian hierarchical modeling. The Bayesian approach allows a probabilistic statement like:

“There’s an 80% chance the peptide raised testosterone by more than 25%.”

That 0.80 probability threshold serves as the decision point for declaring a pharmacodynamic effect.

Why This Matters

If VDAC1-targeting peptides can safely increase testosterone without suppressing the body’s own production, they could represent a new category of precision endocrine therapy; one that enhances hormone output from within rather than replacing it externally.

Instead of injecting testosterone and shutting down natural production, these peptides might allow a gentler, more physiological boost.

What’s Next

This experiment is a first step. If clear effects appear, the next phase could include:

• A small series of replicated N-of-1 trials (5–10 participants)

• Bayesian synthesis of results across individuals

• A follow-up dose-ranging study

• Eventually, a small randomized crossover comparing peptides to low-dose testosterone therapy or SERMs

All data and analysis code will be released publicly once complete to ensure transparency and reproducibility.

Final Thought

This project explores a new angle on hormone optimization: not by overwhelming the body with hormones, but by fine-tuning the machinery that makes them. It’s a bottom-up approach to restoring balance, starting from the mitochondria outward.

See below for the full experimental outline and protocol. Peptide synthesis and supply was provided by Upgraded Bio (admin@upgraded.bio).

Experiment Planning

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