**Extended Data Fig. 9** | Fasting increases hypusination of eIF5A. (a) Hypusine levels are increased after 24 hours -N in yeast BY4741 GFP-Atg8 cells, but not in Δspe1. Representative immunoblot. (b) Quantification of [A]. N = 7 (Δspe1-N), 8 (rest) biologically independent samples (yeast cultures). (c) Representative immunoblot of WT and Δspe1, with and without 100 µM SPD after 6 hours -N, assessed for hypusine and GAPDH. (d) Quantification of [C]. N = 6 biologically independent samples (yeast cultures). (e) SPD supplementation does not further increase hypusination in BY4741 GFP-Atg8 yeast. Representative immunoblot. (f) Quantification of [E]. N = 4 biologically independent samples (yeast cultures). (g) Representative maximum projection images of confocal microscopy images of male w<sup>III18</sup> fly central brain regions probed for eIF5A and hypusine by immunofluorescence. Prior to dissection, the flies were fasted for 0 (ad lib) or 12 hours, starting at 8 pm. Scale bar = 50 µm. (h) Quantification of signal intensities as shown in [G]. N = 30 fly brains. (i) Representative immunoblot of female Drosophila w<sup>III18</sup> head extracts, assessed for hypusine and actin after 12 and 24 hours overnight fasting. RF = 12 hours refeeding. (j) Quantification of [I]. N = 6 biologically independent samples (fly head lysates). (k) Representative immunostaining microscopy images against hypusine after 12 hours fasting of

female WT *w*<sup>III18</sup> and *Odc1<sup>MI10996</sup>+/+ fly brains. Scale bar = 50 µm. (l) Quantification of [K]. N = 11(ad lib), 12(fasted) fly brains. (m) Immunoblots of cardiac tissue from young male transgenic IGF1R<sup>fl</sup> or dnPI3K mice and their age-matched WT controls. Heart lysates were assessed for hypusine, total eIF5A and GAPDH. (n) Quantification of [M]. N = 6(WT), 7(IGF1R<sup>fl</sup>), 3(dnPI3K) mice. (o) Representative immunoblot of human U2OS cells starved for 6 hours and assessed for hypusine, total eIF5A and GAPDH. (p) Quantification of [O]. N = 6 biologically independent samples. (q) Hypusine levels after five days of fasting in isolated human PBMCs of cohort 4, stratified by sex. N = 12(female baseline), 11(female fasted), 5 (male baseline), 4(male fasted) volunteers. (r-t) eIF5A<sup>fl</sup> levels after five days of fasting in isolated human PBMCs of cohort 4 do not correlate with age, pre-fasting baseline BMI or body weight loss (body weight ratio). N = 15 volunteers. *Statistics*: [B,D,L,NP,Q] Two-way ANOVA with Holm-Šídák's multiple comparisons test. [F] One-way ANOVA with Holm-Šídák's multiple comparisons test. [H,J] Two-tailed Student's t-test with Holm-Šídák's multiple comparisons test. [R-T] Simple linear regression. Bar graphs show the mean ± s.e.m. Source numerical data and unprocessed blots are available in source data.
<caption>A</caption>

C. elegans

<figure><img src="image_116.png" /><figcaption>B</figcaption></figure>

GFP

mCherry

Merge

<figure><img src="image_117.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_118.png" /><figcaption>D</figcaption></figure>

Ad lib.

Fasted

<figure><img src="image_119.png" /><figcaption>E</figcaption></figure>

mCherry/GFP

<figure><img src="image_120.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_121.png" /></figure>

SQST-1::GFP

C. elegans

<caption>G</caption>

SQST-1::GFP

<figure><img src="image_122.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_123.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_124.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_125.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_126.png" /></figure>

Extended Data Fig. 10 | See next page for caption.
**Extended Data Fig. 10** | Hypusination is important for fasting-induced autophagy in worms and human cells. (a) Feeding of *dhps-1(RNAi)* expressing bacteria reduces *dhps-1* mRNA expression in *C. elegans* N2 worms. N = 2 biologically independent experiments. (b) Representative confocal images of the head region of young *C. elegans* MAH215 (sqls11 [lgg-1p::mCherry::GFP::Igg-1+rol-6]) fasted for two days and fed control or *dhps-1(RNAi)* expressing bacteria. Autolysosomes (ALs) appear as mCherry-positive puncta. Scale bar = 50 µm. (c) Quantification of ALs as depicted in [B]. Note that the experiment and statistics were performed together with the *odc-1(RNAi)* groups in Fig. 3p. N = 11(CTL ad lib), 26(CTL fasted), 10(*dhps-1(RNAi)* ad lib), 21(*dhps-1(RNAi)* fasted) worms. (d) Representative fluorescence images of the head region of young *C. elegans* MAH215 (sqls11 [lgg-1p::mCherry::GFP::Igg-1+rol-6]) (LGG-1 is the *C. elegans* ortholog of LGG-1/Atg8) fasted for two days and fed control or *dhps-1(RNAi)* expressing bacteria. Autophagic activity is indicated by a shift to the red spectrum due to fluorescence quenching of the pH-sensitive-GFP by the acidic environment of the autolysosome. Scale bar = 50 µm. (e) Quantification of the ratio of the mean fluorescence intensity of mCherry/GFP signals, as depicted in [D]. Note that the experiment and statistics were performed together with the *odc-1(RNAi)* groups in Extended Data Fig. 6d. N = 121(CTL ad lib), 127(CTL fasted),

107(*Dhps-1(RNAi)* ad lib), 143(*Dhps-1(RNAi)* fasted) worms. (f) Representative fluorescence images of the head region of young *C. elegans* SQST-1::GFP fasted for two days and fed control or *dhps-1(RNAi)* expressing bacteria. Autophagic activity is indicated by a decrease in the number of GFP-positive particles. Scale bar = 50 µm. (g) Quantification of the SQST-1::GFP particles in the head region, as depicted in [F]. Note that the experiment and statistics were performed together with the *odc-1(RNAi)* groups in Extended Data Fig. 6f. N = 133(CTL ad lib), 86(CTL fasted), 108(*dhps-1(RNAi)* ad lib), 100(*dhps-1(RNAi)* fasted) worms. (h) Knockdown of *dhps-1* does not affect worm size under ad lib or IF conditions. Note that the experiment and statistics were performed together with the *odc-1(RNAi)* groups in Extended Data Fig. 7g. N = 56(CTL ad lib), 66(CTL IF), 62(*dhps-1(RNAi)* ad lib), 59(*dhps-1(RNAi)* IF) worms. (i) Representative images of U2OS GFP-LC3 cells starved in HBSS with or without 100 µM GC7 for 6 hours. CQ was added for 3 hours before fixation. (j-k) Quantifications of [I]. N = 18 biologically independent samples. *Statistics*: [C,E,G] Kruskal-Wallis-test with Dunn's correction. [H,J,K] Two-way ANOVA with Holm-Šidák's multiple comparisons test. Bar graphs show the mean ± s.e.m. Source numerical data are available in source data.
## Reporting Summary

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## Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

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## Software and code

Policy information about [availability of computer code](#)

Data collection MetaXpress (Molecular Devices), Image Lab (BD), FACSDiva (BD),

Data analysis MetaXpress (Molecular Devices), ImageJ (2.3.0), Graphpad Prism 10.1.0, Metaboanalyst 5.0, Chenomx NMR Suite 8.4 (Chenomx Inc.), Matlab 2014b, Microsoft Excel for Mac and Windows (Version 16.84), ImageLab 5.2 (Bio-Rad), DIA-NN (v1.8.0), Xcalibur v4.0 and v2.2 Software (Thermo Fisher Scientific).

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## Data

Policy information about [availability of data](#)

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The mass spectrometry yeast proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (http://www.ebi.ac.uk/pride) partner
repository with the dataset identifier PXD035909. The biological material and datasets, including NMR and MS metabolomics data, generated and/or analyzed during the current study are in the source data or available from the corresponding authors on reasonable request.

## Research involving human participants, their data, or biological material

Policy information about studies with **human participants or human data**. See also policy information about **sex, gender (identity/presentation), and sexual orientation** and **race, ethnicity and racism**.

**Reporting on sex and gender**

The main findings of increased spermidine levels and hypusine upon fasting in humans occur irrespective of the participants' sex. Sex was determined by self-reporting and gender data was not collected. Due to the implications of these analyses, we did not perform sex-stratified analyses for the rest of the analyses.

**Reporting on race, ethnicity, or other socially relevant groupings**

We only used self-reported sex as a category. We did not use other categories or factors in our analyses of human samples. All other details are listed in Table S1 and the respective publications.

**Population characteristics**

Population characteristics for each cohort are listed in Table S1 and the respective publications.

**Recruitment**

Recruitment strategies are listed in the specific methods section of each cohort and/or in the respective publications. Selection bias is possible due to the participant's self-interest in fasting interventions.

**Ethics oversight**

As stated in the Materials & Methods section. Medical council of Baden-Württemberg, Germany; Ethics Committee of the Charité-University Medical Center Berlin, Germany; Ethics committee of the Medical University of Graz, Austria; Institutional review board of Charité Universitätsmedizin Berlin, Germany

Note that full information on the approval of the study protocol must also be provided in the manuscript.

## Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_127.png" /></figure>

Life sciences

<figure><img src="image_128.png" /></figure>

Behavioural & social sciences

<figure><img src="image_129.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

## Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

**Sample size**

Sample sizes were based on common and feasible sample sizes used in the respective field of research, without performing power analysis. Metabolite measurements of human samples relied on pre-established cohorts, that were collected for primary endpoints as described in the respective publication, study protocol or clinical registry entry (see Table S1).

**Data exclusions**

Outlier analysis by the ROUT method using Graphpad Prism 10.1.0 and outlier exclusion were performed exclusively on the metabolite measurements of human biological samples. Data exclusion criteria were not pre-established.

**Replication**

Appropriate numbers of independent biological samples and independent experimental repeats were used in all experiments, varying based on the model organism and technical requirements. All data points shown are independent samples. All experimental replications yielded comparable outcomes.

**Randomization**

Allocation to the experimental groups was random. Randomization procedures for human participants are described in detail in the respective publications and study protocols (see Table S1).

**Blinding**

Worms, flies and mice were randomly assigned to the experimental groups, but the experimenters were not blinded, except for the measurement of mouse healthspan (echocardiography, frailty index, body composition, wire hanging capability, grip strength, body surface temperature, etc.). During continuous (intermittent) fasting experiments it is not possible to blind the person handling the experiment due to obvious reasons. Also, the manual assessment of yeast GFP-Atg8 microscopy was performed in a blinded fashion. All other experiments were conducted in a non-blinded manner.

## Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
## Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☐</td><td>Antibodies</td></tr><tr><td>☐</td><td>Eukaryotic cell lines</td></tr><tr><td>☒</td><td>Palaeontology and archaeology</td></tr><tr><td>☐</td><td>Animals and other organisms</td></tr><tr><td>☐</td><td>Clinical data</td></tr><tr><td>☒</td><td>Dual use research of concern</td></tr><tr><td>☒</td><td>Plants</td></tr></tbody></table>

## Methods

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☒</td><td>ChIP-seq</td></tr><tr><td>☒</td><td>Flow cytometry</td></tr><tr><td>☒</td><td>MRI-based neuroimaging</td></tr></tbody></table>

## Antibodies

**Antibodies used**

anti-GFP (Roche #1814460), anti-hypusine (Merck #ABS1064-I), anti-eIF5A (BD #611977), anti-GAPDH clone GA1R (Thermo Fisher Scientific #MA5-15738), anti-HA (Sigma #H 9658), anti-mouse IgG (Sigma #A9044), anti-rabbit IgG (Sigma #A0545), Sch9-pThr737 (custom), anti-Sch9 (custom), anti-Adh1 (Calbiochem, #126745), HRP-linked anti-rabbit IgG (Sigma #A0545), anti-beta actin (abcam #ab197277), anti-rabbit Cy5 (Invitrogen), goat anti-guinea pig Alexa 555 (Invitrogen)

**Validation**

Antibodies were validated by the manufacturers or internally by protein-lowering strategies (e.g. pharmacological inhibition of hypusination for anti-hypusine)

## Eukaryotic cell lines

Policy information about cell lines and Sex and Gender in Research

**Cell line source(s)**

human glioblastoma H4 (ATCC), human osteosarcoma U2OS (ATCC)

**Authentication**

Cell lines were not authenticated.

**Mycoplasma contamination**

Cell lines were mycoplasma-free.

**Commonly misidentified lines**

(See ICLAC register)

No commonly misidentified lines were used in this study.

## Animals and other research organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

**Laboratory animals**

male and female 8-12 weeks old BALB/CjRj mice (Janvier Labs); 4, 9, 16-17 and 20 months old C57BL/6J:Rj mice (Janvier Labs); transgenic male IGF1Rtg mice and male dnPI3K mice up to 9 months of age; serum of transgenic K/BxN mice; male and female w1118 Drosophila melanogaster at varying ages across their lifespan; male and female heterozygous Odc1MI10996/+ loss-of-function mutant flies (BDSC #56103) at varying ages across their lifespan; male and female heterozygous eIF5AK51R/+ flies at varying ages across their lifespan;

**Wild animals**

The study did not involve wild animals.

**Reporting on sex**

Since sex did not have an impact on spermidine levels during fasting in humans, we did not consider sex in the pre-clinical study design for all analyses. The sex of the animals was explicitly stated in the figure legends, Materials & Methods section and the main text. We used male and female mice for metabolomics (separately). The investigation of the cardioprotective and healthspan-promoting effects of intermittent fasting were performed on male mice only. We used a 1:1 mixed-sex cohort for the investigation of intermittent fasting on an arthritis model, analysed together and per sex.

**Field-collected samples**

The study did not involve samples collected from the field.

**Ethics oversight**

All animal experiments were performed in accordance with national and European ethical regulation (Directive 2010/63/EU) and approved by the responsible government agencies (Bundesministerium für Wissenschaft, Forschung und Wirtschaft, BMWFW; BMWFW-66.007/0029-WF/V/3b/2017, GZ 2021-0.524.242, GZ 2022-0.137.213, BMWFW-66.010/0160-WF/V/3b/2014, BMWFW-66.010/0198-WF/V/3b/2017, BMBWF-66.010/0042-V/3b/2018). Animal studies using protocol 3 (IF24:24) and the arthritis model were approved by the local ethical committee (District Government of Lower Franconia, #55.2-2532-2-1041-15). All experiments were conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). Calorie-restricted mice (protocol 4) were used in accordance with protocols approved by the Institutional Animal Care and Use Committee of the NIA.

Note that full information on the approval of the study protocol must also be provided in the manuscript.
## Clinical data

Policy information about clinical studies

All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

**Clinical trial registration** DRKS00016657, DRKS00010111 (German Clinical Trials Register), NCT02673515, NCT 04739852 (Clinicaltrials.gov)

**Study protocol** Published study protocols are listed in Table S1, where available. For the rest we refer to the clinical trial registries and previous publications.

**Data collection** Details of recruitment strategies and data collection are described in the referenced publications and clinical trial registry entries.

**Outcomes** None of the herein described metabolite measurements are part of primary endpoints in the respective trials. Primary endpoints of these trials have been published elsewhere (Table S1 summarizes previous publications).

## Plants

**Seed stocks** Report on the source of all seed stocks or other plant material used. If applicable, state the seed stock centre and catalogue number. If plant specimens were collected from the field, describe the collection location, date and sampling procedures.

**Novel plant genotypes** Describe the methods by which all novel plant genotypes were produced. This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization. For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed. For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

**Authentication** Describe any authentication procedures for each seed stock used or novel genotype generated. Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g. second site T-DNA insertions,mosiacism, off-target gene editing) were examined.