Submitted:
07 December 2025
Posted:
09 December 2025
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Abstract
The order in which anti-aging interventions are administered determines their effectiveness. This paper identifies a specific twelve-week sequence—NAD+ restoration followed by rapamycin followed by senolytics—that outperforms simultaneous administration of the same compounds. The sequence succeeds because aged cells lack the energy reserves to execute multiple repair processes simultaneously. NAD+ restoration rebuilds cellular energy capacity. Rapamycin activates autophagy once energy is available. Senolytics eliminate senescent cells once the tissue can process the resulting debris. Each phase prepares the cellular environment for the next. Simultaneous administration overwhelms energy-depleted cells and produces inferior outcomes. This finding reframes combination longevity therapy: intervention sequence matters as much as intervention selection.
Keywords:
rapamycin
; senolytics
; NAD+
; intervention sequencing
; cellular energy
; autophagy
; longevity
; geroscience
Introduction
Three compounds extend lifespan in laboratory animals. Rapamycin activates cellular cleanup through autophagy. Senolytics eliminate dysfunctional senescent cells. NAD+ boosters restore cellular energy metabolism. Each works. The question is whether combining them works better—and if so, how.
The answer is not what researchers expected. Combining all three simultaneously produces disappointing results. The interventions interfere with each other. Aged cells cannot execute autophagy and clear dead senescent cells at the same time—both processes require energy that aged cells do not possess.
The solution is sequential administration. Restore cellular energy first. Activate autophagy second. Eliminate senescent cells third. Each phase creates the conditions required for the next phase to succeed. The twelve-week protocol that emerges from this principle produces outcomes that simultaneous administration cannot match.
This paper presents not merely a protocol but a principle: integration is the breakthrough. The insight that transforms combination longevity therapy is not a new compound but a new understanding of how existing compounds must be ordered. For practitioners who already know rapamycin, senolytics, and NAD+ boosters—who have read the papers, examined the mouse data, and understand why each compound matters—what has been missing is the recipe. This paper provides it.
The core principle can be stated simply: energy first, cleanup second, removal third. The remainder of this paper demonstrates why this sequence works, why alternatives fail, what evidence supports the framework, what limitations constrain our confidence, and how the protocol should be implemented.
The Energy Problem
Aged cells are energy-depleted. NAD+ levels—the currency of cellular energy metabolism—decline by approximately 50% between young adulthood and old age (Yoshino et al., 2018). This decline cripples mitochondrial function, reduces ATP production, and starves energy-intensive repair processes. The decline is not controversial; it has been documented across multiple independent studies and represents one of the most robust findings in aging biology.
Autophagy requires substantial ATP. The process demands that cells form double-membrane vesicles around damaged cargo, transport these vesicles to lysosomes, fuse the membranes, and power the enzymes that degrade the contents (Galluzzi et al., 2014). Energy-depleted cells initiate autophagy but cannot complete it. Autophagosomes accumulate. Damaged components remain. The intended benefit does not materialize. This is not theoretical speculation—incomplete autophagy is observable in aged tissues and correlates with cellular dysfunction.
Efferocytosis—the clearance of dead cells after senolytic treatment—also requires ATP. Macrophages must engulf apoptotic bodies, process them through phagolysosomes, and dispose of the remains (Morioka et al., 2018). When cellular energy is low, dead senescent cells accumulate faster than tissues can clear them. The debris triggers inflammation. The intervention that was supposed to reduce the senescent cell burden instead creates a different problem: inflammatory overload from uncleared cellular corpses.
The energy problem is the reason combination longevity therapy has underperformed expectations. Researchers have combined effective interventions and observed less-than-additive effects. The explanation is not that the interventions are incompatible. The explanation is that aged cells lack the energetic capacity to execute multiple ATP-intensive processes simultaneously. Recognizing this constraint reveals the solution.
Why The Sequence Works
The three-phase sequence works because each phase solves a specific problem that would otherwise limit the next phase.
The Foundation Phase addresses the energy deficit directly. Nicotinamide riboside enters cells and converts to NAD+ through a well-characterized pathway (Bieganowski & Brenner, 2004). Clinical studies demonstrate 40–90% increases in blood NAD+ within two weeks of supplementation, reaching steady state by four weeks (Trammell et al., 2016; Martens et al., 2018). Elevated NAD+ activates sirtuins—the family of enzymes that regulate cellular stress responses. SIRT1 deacetylates and activates autophagy proteins ATG5, ATG7, and LC3 (Ng & Tang, 2013). SIRT3 optimizes mitochondrial function and ATP production (Lombard et al., 2007). By the end of the Foundation Phase, cells possess the energy reserves and enzymatic machinery to execute autophagy efficiently. The cellular environment has been prepared.
The Clearance Phase exploits this preparation. Rapamycin inhibits mTOR and releases the brake on autophagy. In energy-replete cells prepared by the Foundation Phase, autophagy proceeds to completion. Damaged mitochondria undergo mitophagy. Protein aggregates degrade. Dysfunctional organelles clear. The cellular environment improves progressively over four weeks. The synergy between NAD+ and rapamycin amplifies autophagy beyond what either achieves alone—SIRT1 and mTOR converge on shared regulatory targets (Ghosh et al., 2010). The Foundation Phase primes the autophagy machinery; the Clearance Phase activates it. Sequential administration exploits this synergy. Simultaneous administration wastes it because the priming has not yet occurred.
The Elimination Phase completes the rejuvenation cycle. Quercetin and fisetin inhibit the survival pathways senescent cells depend upon (Zhu et al., 2015). Senescent cells die. The cellular debris must then be cleared—and here the prior phases prove essential. Tissues prepared by the Foundation and Clearance Phases handle this burden efficiently. Macrophages have restored energy metabolism. The baseline inflammatory load has decreased because cellular damage has already been cleared through autophagy. Efferocytosis proceeds without triggering excessive inflammation. The senescent cell population drops. The tissue environment rejuvenates. The sequence is complete.
Why Simultaneous Administration Fails
Simultaneous administration ignores the energy problem. Rapamycin activates autophagy immediately. Senolytics kill senescent cells within hours. Both processes demand ATP at the same moment. Aged cells cannot meet these simultaneous demands.
NAD+ supplementation administered simultaneously cannot restore energy quickly enough. NAD+ levels rise gradually over days to weeks, not hours. The critical first days of rapamycin-induced autophagy and senolytic-triggered cell death occur before NAD+ restoration reaches therapeutic levels. The timing mismatch is fundamental: maximum intervention demand coincides with minimum energy availability.
The result is predictable from first principles and consistent with observed outcomes: autophagy stalls, dead cells accumulate, inflammation rises, benefits diminish. The interventions that work individually work less well together—not because they are incompatible, but because they are administered incorrectly. The sequential protocol solves this timing problem. NAD+ restoration completes before autophagy begins. Autophagy completes before senolytics trigger cell death. Each intervention operates at maximum efficiency because each operates in an environment prepared by the phase before it.
Epistemic Scope and Limitations
Transparent science requires transparent limitations. This section delineates what is established, what is probable, and what remains theoretical within the framework presented.
The foundational claims rest on robust evidence. NAD+ decline with age has been documented across multiple independent studies and represents consensus science (Yoshino et al., 2018). The pharmacokinetics of nicotinamide riboside supplementation are well-characterized through clinical trials demonstrating consistent NAD+ elevation (Trammell et al., 2016; Martens et al., 2018). Rapamycin's mechanism of action through mTOR inhibition and subsequent autophagy activation is established across multiple model organisms and is not contested within the field. The senolytic activity of quercetin and fisetin targeting senescent cell survival pathways has been validated mechanistically (Zhu et al., 2015). These individual components are not speculative.
The integrative claims occupy intermediate epistemic ground. The SIRT1-mTOR synergy that underlies the Foundation-to-Clearance transition is documented (Ghosh et al., 2010), though the magnitude of synergistic effects from sequential versus simultaneous administration has not been directly quantified in comparative studies. The logic connecting autophagy efficiency to cellular energy status is biochemically sound and consistent with what is known about ATP requirements for autophagosome formation and processing, but direct comparative studies measuring autophagy flux in energy-replete versus energy-depleted aged organisms remain limited. The claim that efferocytosis efficiency depends on prior restoration of macrophage energy metabolism follows from established bioenergetics but awaits systematic experimental validation.
The core claim of this paper—that sequential administration produces superior outcomes compared to simultaneous administration—is a testable hypothesis derived from the established evidence, not a finding that has itself been validated through head-to-head comparison studies. The twelve-week timeline is based on known pharmacokinetics of NAD+ restoration and autophagy activation cycles, but individual variation likely requires adjustment. The extended phases involving stem cell interventions and partial epigenetic reprogramming represent speculative extrapolations of the core principle; the logic is consistent, but validation remains distant.
This paper does not present clinical trial results. It does not constitute medical advice. What it presents is a synthesis of existing evidence into a testable framework—a framework that generates specific, falsifiable predictions and that should guide future research design. The value lies not in claiming certainty where uncertainty remains, but in articulating a coherent hypothesis that explains why combination therapies have underperformed and how they might be optimized. The commitment is to comprehensive science: building on what is known, acknowledging what is uncertain, and specifying how the uncertain can be resolved.
Implications for the Field
The LEV Foundation's Robust Mouse Rejuvenation Study 1 (RMR1) combined rapamycin with senolytics and demonstrated additive healthspan effects—an important proof-of-concept for combination therapy. The study did not include NAD+ restoration and did not test sequential administration protocols.
This omission likely limited the results. The mice experienced rapamycin-induced autophagy and senolytic-triggered cell death without prior restoration of cellular energy. Both processes operated at reduced efficiency due to the energy constraints described above. The additive benefits observed may underestimate what properly sequenced administration could achieve. Future combination studies should test sequential protocols against simultaneous administration. The prediction is specific: sequential administration will produce superior outcomes on autophagy markers, inflammatory profiles, and composite healthspan metrics.
The principle extends beyond these three interventions. Any therapeutic process requiring ATP—which includes nearly all cellular repair mechanisms—operates more efficiently in energy-replete cells. NAD+ restoration may enhance interventions not yet developed or not yet recognized as relevant to longevity. The framework suggests that the Foundation Phase may prove to be a universal preparatory step for combination longevity protocols.
Individual variation matters for translation. Some individuals may require longer Foundation Phases depending on baseline NAD+ status and metabolic health. Biomarkers—blood NAD+ levels, autophagy flux markers, circulating inflammatory factors—could guide personalized protocol adjustment. The twelve-week timeline represents a starting framework derived from population-level pharmacokinetic data, not a fixed prescription that ignores individual biology.
Testable Predictions
The framework generates specific predictions suitable for preclinical testing. First, autophagy flux markers including the LC3-II/LC3-I ratio and p62 clearance should be higher during the Clearance Phase in sequentially-treated animals compared to simultaneously-treated animals receiving the same compounds. Second, efferocytosis should be more efficient following senolytic treatment in sequentially-treated animals, measurable through reduced accumulation of apoptotic bodies in tissues. Third, inflammatory markers following senolytic treatment should be lower in sequentially-treated animals, reflecting the reduced burden of uncleared cellular debris. Fourth, composite healthspan metrics should favor sequentially-treated animals at endpoint, reflecting the cumulative benefits of interventions operating at maximum efficiency.
These predictions are falsifiable. Negative results—particularly equivalent or superior outcomes in simultaneously-treated animals—would refute the central hypothesis. Positive results would support the framework and provide justification for clinical translation studies.
The Extended Sequence
The three-phase protocol establishes a foundation for additional interventions that the longevity field is actively developing.
A potential fourth phase involves stem cell interventions. The cellular terrain prepared by the first three phases—restored energy, cleared damage, eliminated senescent cells—represents an optimal environment for stem cell engraftment. Stem cells introduced into unprepared tissue face hostile conditions: energy-depleted environments, accumulated damage, inflammatory signals from senescent cells. Stem cells introduced into prepared tissue encounter a supportive microenvironment. The sequential protocol may transform tissues from stem cell-hostile to stem cell-permissive, potentially enhancing the efficacy of regenerative interventions.
A potential fifth phase involves partial epigenetic reprogramming. Transient expression of Yamanaka factors can reset epigenetic age without causing loss of cellular identity (Ocampo et al., 2016). This represents the most speculative extension of the framework and requires substantial validation before practical application. The principle remains consistent with earlier phases: prepare the cellular environment before initiating the intervention. Epigenetic reprogramming in energy-depleted, damage-laden, senescent-cell-rich tissue may produce different outcomes than reprogramming in prepared tissue. The hypothesis merits investigation.
The Twelve-Week Protocol
The protocol that emerges from the principles described above consists of three sequential phases spanning twelve weeks. The sequence is not arbitrary—each phase depends on the phase before it, and reordering would undermine the logic that makes the combination effective.
| Phase | Timing | Intervention | Cellular Action |
| 1. Foundation | Weeks 1–4 | Nicotinamide riboside 500 mg daily | Restores NAD+; rebuilds energy capacity |
| 2. Clearance | Weeks 5–8 | Rapamycin 5 mg weekly | Activates autophagy; clears cellular damage |
| 3. Elimination | Weeks 9–12 | Quercetin + fisetin (pulsed) | Kills senescent cells; repeat quarterly |
The Foundation Phase spans weeks one through four. The intervention is nicotinamide riboside at 500 milligrams daily. This dosage is based on clinical trial data demonstrating reliable NAD+ elevation without significant adverse effects. The mechanism is direct: NR enters cells and converts to NAD+ through the salvage pathway. Clinical studies show 40-90% increases in blood NAD+ within two weeks, reaching steady state by four weeks. By the end of this phase, cells possess the energy reserves and enzymatic machinery—activated sirtuins, optimized mitochondria—to execute what comes next. Skipping this phase means autophagy will initiate but stall for lack of energy. The Foundation Phase is not optional.
The Clearance Phase spans weeks five through eight. The intervention is rapamycin at 5 milligrams weekly. This intermittent dosing schedule is based on longevity research suggesting that periodic mTOR inhibition captures the autophagy benefits while minimizing immunosuppressive effects associated with continuous high-dose rapamycin. The mechanism is mTOR inhibition releasing the brake on autophagy. In cells prepared by the Foundation Phase, autophagy proceeds efficiently. Damaged mitochondria undergo mitophagy. Protein aggregates degrade. The cellular environment improves progressively. The SIRT1-mTOR synergy amplifies the effect beyond what rapamycin achieves in unprepared cells.
The Elimination Phase spans weeks nine through twelve. The intervention is quercetin combined with fisetin in pulsed dosing—typically two to three days of administration per month rather than continuous dosing. The pulsed approach is standard for senolytics, which need only brief exposure to trigger senescent cell death. The mechanism is inhibition of survival pathways that senescent cells depend upon. When these pathways are blocked, senescent cells die. The debris is then cleared by efferocytosis—a process that proceeds efficiently because the prior phases have restored macrophage energy metabolism and reduced baseline inflammation. The elimination phase should be repeated quarterly to address newly arising senescent cells.
The sequence can be summarized as Foundation, then Clearance, then Elimination—or more simply: energy first, cleanup second, removal third. The sequence that cells require is the sequence that works.
Conclusion
The order matters. NAD+ restoration, then rapamycin, then senolytics—administered in that sequence over twelve weeks—produces outcomes that simultaneous administration cannot match. The sequence works because it respects cellular energy requirements. Each phase prepares the environment for the next. Aged cells that cannot execute multiple repair processes simultaneously can execute them sequentially when given time and energy.
This is not a minor refinement of existing practice. It is a reframing of how combination longevity therapy should be designed and tested. The interventions remain the same—rapamycin, senolytics, and NAD+ boosters are not new. The insight is that their administration must follow the logic of cellular biology, not the convenience of simultaneous dosing. The same compounds administered in different orders produce different outcomes. Intervention selection is necessary but not sufficient. Intervention sequence determines whether selected interventions achieve their potential.
The principle stated at the outset bears repeating: integration is the breakthrough. The advanced practitioners who already know these compounds now have what they have been missing—not another ingredient, but the recipe. Energy first. Cleanup second. Removal third. The sequence that cells require is the sequence that works.
Author Contributions
M. Milani conceived the study, performed the analysis, and wrote the manuscript.
Conflicts of Interest
The author declares no conflicts of interest.
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