VITAL: Vaccination, Immunity, Time-restricted Eating, Aging and Lifestyle

Change in autophagic flux in peripheral immune cells from baseline (V1) to four weeks (V2), comparing the TRE group versus control, as measured by flow cytometry detection of LC3-II accumulation via antibody staining.

Change in body weight

Change in body composition (e.g., fat and lean body mass), assessed via Air Displacement Plethysmography (ADP, BodPod).

Change in body composition (e.g., fat and lean body mass), assessed via bioimpedance analysis (BIA).

Change in the differential blood count, assessed with Hematology analyzers.

Change in blood glucose levels, assessed via routine blood diagnostic tests

Changes in insulin metabolism parameters, including insulin, IGF-1, C-peptide, ghrelin, leptin, adiponectin, and glucagon, assessed via ELISAs

Changes in blood ketone body levels (3-hydroxybutyrate, acetoacetate, acetone), assessed using commercially available assay kits

Changes in blood lipids assessed via metabolomics and routine blood diagnostic tests

Changes in blood parameters of liver and renal function assessed via routine blood diagnostic tests

Changes in blood electrolyte levels assessed via routine blood diagnostic tests

Change in the incidence of clinically diagnosable infections during the study period, assessed at study visits by the study physician

Change in continuous blood glucose levels, assessed with continuous glucose monitors (CGM). CGMs will be applied at V1 and V2 and record continuous glucose levels for 14 days each.

Change in grip strength, assessed with a dynamometer. Handgrip strength will be measured using a dynamometer three times sequentially and repeated after 1 hour.

Change in metabolic rate via indirect calorimetry. Indirect calorimetry will be performed to assess energy expenditure as well as carbohydrate and fat oxidation rates. During the procedure, the subject will rest quietly in bed while wearing a ventilated hood. The hood is equipped with an inlet and an outlet valve. Through the inlet valve, the subject receives a constant supply of fresh air, while exhaled air is collected via the outlet valve. Sampling at the rear of the calorimetry device allows determination of gas concentrations in the ambient room air. By calculating the difference, oxygen consumption (VO₂) and carbon dioxide production (VCO₂) in ml/min are determined. These volumes enable the calculation of energy expenditure and substrate oxidation rates. This measurement is performed after a rest period of 30 minutes to determine resting fasting energy expenditure (duration approximately 30 minutes).

Change in Sleep Quality assessed using a questionnaire for the Pittsburgh Sleep Quality Index [PSQI].

Change in EQ-5D-5L assessed using questionnaires.

Change in ChronoType assessed using the Munich ChronoType Questionnaire [MCTQ]

Change in the MPI for geriatric assessment. MPI assessments will be conducted, including documentation of medication and dietary supplement use, medication allergies, nutritional therapies, a range of disease symptoms, social history, activities of daily living (ADL), instrumental activities of daily living (IADL), malnutrition screening, the Short Portable Mental Status Questionnaire (SPMSQ), and the Exton Smith Scale (assessment of decubitus risk), all administered via standardized questionnaires.

A 10-meter walk test will be performed to determine walking speed in m/s. For this purpose, the subject will walk from a 0-meter mark to a 10-meter mark upon command. Timing will begin at the 2-meter mark. The procedure will be repeated three times, and the times will be averaged. If the subject's fitness level permits, the procedure will be repeated with the instruction to complete the 10 meters 'as fast as possible while still feeling safe'.

Change in proteome profiles in plasma and PBMCs, measured by LC-MS mass spectrometry or Olink proteomics.

Change in metabolite patterns in plasma, assessed by untargeted (¹H-NMR) and/or targeted (LC-MS) metabolomics.

Change in metabolite patterns in stool, assessed by untargeted (¹H-NMR) and/or targeted (LC-MS) metabolomics.

Change in metabolite patterns in urine, assessed by untargeted (¹H-NMR) and/or targeted (LC-MS) metabolomics.

Change in gut microbiome composition by metagenomic sequencing of stool.

Change in immune-cell subset distribution and activation, assessed by scRNA-seq and flow cytometry.

Change in body fat percentage, measured by air-displacement plethysmography.

Change in office systolic and diastolic blood pressure (mmHg), measured by non-invasive blood pressure cuff.

Change in pulse-wave velocity (m/s), measured by PulsePen.

Change in frequency of pro- and anti-inflammatory immune cells in PBMCs, by flow cytometric immunophenotyping.

Change in immune-cell function (cytokine production, proliferation) after in vitro stimulation of PBMC subsets, by flow cytometry.

Change in circulating pro- and anti-inflammatory cytokine concentrations in plasma, by multiplex cytokine assay.

Change in expression of senescence markers in immune-cell subsets, by flow cytometry.

Change in the hypusination status of eIF5A in PBMCs by flow cytometry.

Change in mTOR activity status in PBMCs by flow cytometry.

Change in expression of key autophagy-regulatory genes in specific immune-cell types or bulk PBMCs, by qPCR, scRNA-seq, or bulk RNA-seq.

Change in cellular energy metabolism in immune-cell subsets, measured by Seahorse extracellular flux analysis and/or Scenith Assay in isolated immune cells ex vivo.

Change in coagulation, assessed via e.g., thrombin generation, d-dimers from plasma

Change in influenza-specific B- and T-cell responses in serum and PBMCs (e.g., ELISpot).

Change in SARS-CoV-2 specific B- and T-cell responses in serum and PBMCs (e.g., ELISpot).

Change in concentration of influenza-specific IgG antibodies, by ELISA.

Change in concentration of SARS-CoV-2 specific IgG antibodies, by ELISA.

Change in concentration of influenza-neutralizing antibodies.

Change in concentration of SARS-CoV-2 neutralizing antibodies.

Change in DNA-break profiles in primary B cells, assessed by SWIBRID (Switch-joint Breakpoint Repertoire Identification).