Frailty is a geriatric syndrome characterized by reduced physiological reserves and increased vulnerability to stressors. Given its complex phenotypes and underlying biology, robust multidimensional biomarkers are needed to advance personalized care. We aimed to identify serum metabolomics signatures associated with frailty phenotypes and related features. We analyzed serum metabolomics data in 901 participants (47.5

This study investigated the effect of partially substituting dietary animal with plant protein (PP) sources on the fecal microbiota composition and metabolome in men with increased cardiometabolic risk. In a randomized, controlled, crossover feeding trial (NCT04236518), 19 men with high plasma triglycerides and waist circumference completed two 4-week isoenergetic diets: a flexitarian diet high in PP sources (FLEX, 64% PP) and a more animal-based control diet (CON, 36% PP). Fecal microbiota (shotgun metagenomics: taxa and metabolic pathways) and metabolome (targeted LC-MS) profiles were assessed before and after each diet and integrated with the host plasma metabolome. Delta values (Δd28-d1) were computed (n = 15 participants with all samples available), inter-individual variation was extracted to account for cross-over design, and OPLS-DA analyses comparing FLEX and CON Δd28-d1 were performed. Variables were selected based on their contribution to the diet discrimination effect (VIP > 1.5) and significant differences between groups (p-value < 0.05 from the paired Wilcoxon signed-rank test). The gut microbiota diversity remained unchanged, but FLEX reduced taxa associated with animal-based diets (e.g., Alistipes putredinis). Compared to CON, FLEX increased fecal xanthurenic acid and decreased the genetic potential for indole production. Combined with previously reported plasma changes (increased indole propionic acid and decreased indoxyl sulfate after FLEX), these findings suggest a shift away from indole production toward kynurenine and indole propionic acid-related tryptophan pathways, possibly driven by higher fiber intake, particularly from legumes. A one-month flexitarian diet thus modulated in men specific microbial taxa and metabolism, particularly tryptophan catabolism. These coordinated changes in microbial composition, functional potential, and metabolites indicate that diets higher in PP sources influence gut microbiota activities relevant to cardiometabolic health.

Postprandial metabolism plays a key role in cardiometabolic health, and its impairment with ageing is associated with increased disease risk. While yoghurt consumption has been linked to improved fasting metabolic markers, its acute postprandial effects are less well understood, particularly in older adults. This study investigated whether yoghurt consumption influences age-related postprandial metabolic dysregulation. In a randomised crossover design, 14 young (20–35 years) and 14 older (65–80 years) healthy men consumed 600 mL of whole milk or yoghurt following an overnight fast. Biochemical markers (glucose, insulin, triglycerides, TNF-α, IL-6, GIP and ghrelin) were measured at baseline and up to six hours postprandially. Differences in lipid metabolism by age were further investigated by assessing free fatty acid (FFA) responses in the yoghurt phase only. Postprandial responses were analysed for time, age, product and interaction effects, and summarised using incremental area under the curve (iAUC) and incremental maximum concentration (iCmax). Glucose and insulin responses were influenced by product (time × product, p < 0.05), with yoghurt resulting in significantly lower iCmax values compared with milk. In contrast, triglyceride responses were influenced by age (time × age, p < 0.05), with older adults exhibiting higher iAUC and iCmax, delayed peak concentrations and slower return to baseline levels, independently of the product consumed. No significant age × product or age × product × time interactions were observed for any biochemical marker. Among the 37 FFAs quantified in the yoghurt phase, seven – predominantly saturated and abundant in yoghurt – exhibited a significant time × age interaction, accompanied by higher iAUC or iCmax in older adults. Dairy fermentation improved postprandial glucose and insulin responses, whereas ageing predominantly affected lipid dynamics. Fermentation did not attenuate impairments in postprandial triglyceride metabolism in this acute setting. Profiling individual postprandial FFAs may enhance understanding of metabolic flexibility and inform personalised dietary strategies across the lifespan.

BACKGROUND & AIM:Dietary shifts replacing animal protein (AP) with plant protein (PP) sources have been associated with lowering cardiometabolic risk (CMR), but underlying mechanisms are poorly characterized. This nutritional intervention aims to characterize the metabolic changes induced by diets containing different proportions of AP and PP sources in males at CMR. DESIGN:This study is a 4-week, crossover, randomized, controlled-feeding trial in which 19 males with CMR followed two diets providing either 36 % for the control diet (CON-D) or 64 % for the flexitarian diet (FLEX-D) of total protein intake from PP sources. Plasma nontargeted metabolomes (LC-MS method) were measured in the fasted state and after a high-fat challenge meal at the end of each intervention arm. Lipogenesis and protein synthesis fluxes, flow-mediated dilatation (FMD) and gluco-lipidic responses were assessed after the challenge meal. Data were analyzed with mixed models, and univariate and multivariate models for metabolomics data. RESULTS:In both arms CMR improved with time, with decreased body weight (-0.9 %), insulin resistant (-34 %, HOMA-IR, Homeostatic Model Assessment for Insulin Resistance) and low-density lipoproteins (LDL)-cholesterol (-11 %). Diet had no effect on FMD or metabolic fluxes, but a trend (0.05 <p ≤ 0.1) was observed for a stronger decrease in HOMA-IR and lower postprandial glucose after FLEX-D vs CON-D. The abundance of 21 and 37 metabolites differed between diets at fasted and fed states, respectively, including food intake biomarkers of AP (methylhistidine, eicosapentaenoic acid, hydroxyprolines) and PP sources (trigonelline, N-acetyl-ornithine). In fasted or fed states, indole acrylic acid and indole propionic acid, both products of tryptophan catabolism, were higher after FLEX-D vs CON-D, while the indispensable amino acids-related metabolites alpha-aminoadipic acid, hydroxymethylbutyric acids and propionylcarnitine were lower. In the postprandial state only, the ω-oxidation products dodecanedioic, tetradecanedioic and hexadecanedioic acids were higher after FLEX-D vs CON-D. CONCLUSIONS:Despite little changes in risk factors after 4 wk, this study evidenced subtle metabolic adaptations in amino acids and lipid metabolism and gut microbiota activity occurring after higher PP source intake that may be beneficial to CMR. CLINICALTRIALS: GOV STUDY IDENTIFIER:NCT04236518. CLINICAL TRIAL REGISTRY:NCT04236518 on ClinicalTrials.gov.

There is a high inter-individual variability in response to food, determined by multiple interacting factors, such as age, sex, genotype, gut microbiota, eating behaviours, physical activity or socio-demographic factors. Previous studies demonstrated the possibility to predict the postprandial glycemic response to food in healthy adults based on deep phenotyping. We hypothesize that inter-individual variability may be amplified at later ages, as a result of different life trajectories and long-life exposures. The MetabotypAGE project proposes exploring the inter-individual variability in response to food in the elderly (ClinicalTrials.gov Identifier NCT06163794). The first interdisciplinary task aimed to establish the best tools and methods to recruit a large highly diverse group of subjects including those living in rural areas, and to carry out deep phenotyping adapted to the older population living at home. The second objective of MetabotypAGE is an exploratory study on 150 healthy people aged 60 to 75, who will wear a CGM for 2 weeks, during which they will eat four standardized test meals. Their post-prandial glycemia will be followed after the test meals. Furthermore, their metabolic flexibility will be assessed with a nutritional challenge test (type PhenFlex) at the clinical center. Volunteers will be extensively phenotyped with a battery of functional tests (physical aptitude, gustatory, olfactory and masticatory function, cognition, vascular function…), analyses on plasma, PBMC, urine, feces and saliva (biochemical, transcriptomics, metagenomics, and metabolomics) and >30 questionnaires to cover many dimensions including their metabolism, physical capacity, socio-economical status, cognitive function, digestive function, and dietary habits. The volunteers will be classified in various metabotypes using clustering methods, based on the glycemic responses to test meals. Then, the multidimentional data collected will be used (i) to characterize the metabotypes (descriptive statistics) and (ii) to explore links between postprandial response to the test meals and the subjects’ descriptive data, using correlation networks based on a Gaussian Graphical Model method. The MetabotypAGE consortium combines partners with complementary skills in nutrition and health of the elderly, several clinical research structures, and local players in social action for senior citizens. Our ultimate goal is to lay solid bases for the development of tailor-made recommendations for seniors.

We formerly demonstrated that GPR40 prevents from osteoporosis establishment using synthetic agonist. Here, we questioned whether stimulation of GPR40 by fatty acids, the natural ligands for GPR40, may parallel with its described beneficial effects on bone. In this study we demonstrated for the first time that GPR40 limits bone loss induced by ovariectomy upon high fat diet. Taken together, our results demonstrate that GPR40 mediates beneficial effects of high fat diets mainly by targeting the bone cell coupling and subsequent osteoclastic bone resorption.

Nutritional biomarkers of dairy intake can be affected by both food transformation and the metabolic status of the consumer. To assess these effects, this study investigated the serum volatilome of 14 young (YA) and 14 older (OA) adult men undergoing a 3 week restriction of dairy and fermented foods followed by a randomized crossover acute intake of milk and yogurt. 3,5-Dimethyl-octan-2-one was identified as a potential marker of dairy product intake as its response after both milk and yogurt intake was significantly increased during the postprandial phase but significantly decreased in fasting serum samples of the OA group after the restriction phase. The postprandial response of two metabolites was significantly different for the two dairy products while 19 metabolites were modulated by age. Remarkably, the response of all age-dependent metabolites was higher in the OA than in the YA group after milk or yogurt intake, whereas at the end of the restriction phase, their fasting concentrations were lower in the OA than in the YA group. Among these, p-cresol, a specific marker of colonic protein fermentation, had a significant response in the OA but not the YA group, which may suggest impaired intestinal processing of dietary proteins in the OA group.

Background: Plant proteins (PPs) have been associated with better cardiovascular health than animal proteins (APs) in epidemiological studies. However, the underlying metabolic mechanisms remain mostly unknown.Objectives: Using a combination of cutting-edge isotopic methods, we aimed to better characterize the differences in protein and energy metabolisms induced by dietary protein sources (PP compared with AP) in a prudent or western dietary context. Methods: Male Wistar rats (n 1/4 44, 8 wk old) were fed for 4.5 mo with isoproteic diets differing in their protein isolate sources, either AP (100% milk) or PP (50%:50% pea: wheat) and being normal (NFS) or high (HFS) in sucrose (6% or 15% kcal) and saturated fat (7% or 20% kcal), respectively. We measured body weight and composition, hepatic enzyme activities and lipid content, and plasma metabolites. In the intestine, liver, adipose tissues, and skeletal muscles, we concomitantly assessed the extent of amino acid (AA) trafficking using a 15N natural abundance method, the rates of macronutrient routing to dispensable AA using a 13C natural abundance method, and the metabolic fluxes of protein synthesis (PS) and de novo lipogenesis using a 2H labeling method. Data were analyzed using ANOVA and Mixed models.Results: At the whole-body level, PP limited HFS-induced insulin resistance (-27% in HOMA-IR between HFS groups, P < 0.05). In the liver, PP induced lower lipid content (-17%, P < 0.01) and de novo lipogenesis (-24%, P < 0.05). In the different tissues studied, PP induced higher AA transamination accompanied by higher routings of dietary carbohydrates and lipids toward dispensable AA synthesis by glycolysis and beta-oxidation, resulting in similar tissue PS and protein mass.Conclusions: In growing rats, compared with AP, a balanced blend of PP similarly supports protein anabolism while better limiting whole -body and tissue metabolic dysregulations through mechanisms related to their less optimal AA profile for direct channeling to PS.

Background: The effects of supplementation with L-arginine (L-arg), the precursor of nitric oxide (NO), on vascular and cardiometabolic health have largely been explored. Whether other mechanisms of the action of L-arg exist remains unknown, as arginine metabolism is complicated.Objective: We aimed to characterize the effect of low dose L-arg supplementation on overall human metabolism both in a fasting state and in response to an allostatic stress.Methods: In a randomized, double-blind, crossover study, 32 healthy overweight adults (mean age 45 y) with cardiometabolic risk (fasting plasma triglycerides >150 mg/dL; waist circumference >94 cm [male] or >80 cm [female]) were treated with 1.5 g sustained-release L-arg 3 times/d (4.5 g/d) or placebo for 4 wk. On the last day of treatment, volunteers consumed a high-fat meal challenge (900 kcal, 80% as fat, 13% as carbohydrate, and 7% as protein). Plasma was collected at fasting, 2, 4, and 6 h after the challenge, and the metabolome was analyzed by high-resolution liquid chromatography-mass spectrometry. Metabolic profiles were analyzed using linear mixed models-principal component analysis.Results: The challenge meal explained most of the changes in the metabolome. The overall effect of L-arg supplementation significantly explained 0.5% of the total variance, irrespective of the response to the challenge meal (P < 0.05). Among the metabolites that explain most of the L-arg effect, we found many amino acids, including branched-chain amino acids, that were decreased by L-arg supplementation. L-arg also decreased trimethylamine N-oxide (TMAO). Other changes suggest that L-arg increased methyl demand.Conclusions: Analysis of the effect of 4 wk of L-arg supplementation on the metabolome reveals important effects on methyl balance and gut microbiota activity, such as a decrease in TMAO. Further studies are needed to investigate those mechanisms and the implications of these changes for long-term health.

Le vieillissement est marqué par le développement de la fragilité, état clinique multifactoriel conduisant à une dégradation de la capacité d'adaptation des individus aux modifications de leur environnement et de leur autonomie. Pour prévenir et ralentir le syndrome de fragilité et maintenir la qualité de vie à domicile, il est indispensable de détecter précocement les premiers déterminants de cette fragilité afin de mettre en place des stratégies multimodales préventives et efficaces. Parmi les déterminants importants figurent l'état nutritionnel et les capacités fonctionnelles et musculaires. L'objectif notre étude est d'obtenir une signature métabolomique proposant des biomarqueurs précoces d'altération de la mobilité et de l'état nutritionnel, complémentaires de ceux déjà disponibles et, ainsi, d'optimiser l'identification des personnes âgées dont l'état de santé à long terme peut devenir « fragile ». Les échantillons et données prospectives (basal et suivi à 7 ans) de la cohorte Base-II (Demuth at al. 2021) (901 sujets) ont été utilisés. Les critères de fragilité disponibles étaient le score de Fried, deux de ses composantes (la force de préhension de la main (FPM) et le score de dépression) et le statut nutritionnel (Mini Nutritional Assessment [MNA]) en début (T0) et en fin de suivi (T7). Une approche métabolomique par RMN-1H a été réalisée sur les échantillons de sérum prélevés à T0. L'identification et la quantification des métabolites contenus dans ces échantillons ont été effectuées avec le package R ASICS 2.10 (Lefort et al., 2019). La significativité des relations entre les concentrations des métabolites et les différents critères de fragilité a été évaluée par des ANOVA, dans lesquelles les modèles linéaires ont été ajustés par les covariables de temps de suivi, d'âge et de poids à T0. Ces analyses visaient à identifier les métabolites ayant des concentrations : 1) influencées par un effet d'interaction entre la fragilité et le genre (étude sur la population totale) ; et 2) différentes selon les critères de fragilité, pour les hommes et les femmes respectivement (effet genre-dépendent). Les valeurs de p obtenues ont été ajustées pour tenir compte de la multiplicité des tests (procédure de Benjamini et Hochberg), celles inférieures à 5 % ont été considérées comme significatives. Enfin, pour chaque critère de fragilité testé, une analyse d'enrichissement des voies métaboliques (test hypergéométrique, MetaboAnalyst 5.0) a été réalisée sur la liste des métabolites significatifs. Sur les 901 sujets étudiés (428 hommes et 473 femmes), 9 étaient classés comme fragiles, 259 pré-fragiles et 633 robustes (non fragiles) à T0 (respectivement 43, 440 et 418 à T7). La FPM était en moyenne de 34,2 ± 9,2 Newtons à T0 et 27,3 ± 9,7 N à T7. Le MNA a identifié 26 sujets malnutris à T0 et 100 à T7. L'analyse de RMN a identifié et quantifié 83 métabolites. Les modèles en population générale n'ont montré aucun métabolite corrélé aux scores de fragilité, ni aux autres critères évalués. En stratifiant sur le genre, des résultats significatifs ont été trouvés chez les hommes. Ainsi, 27, 31 et 21 métabolites étaient inversement corrélés, avec la FPM à T0 et à T7 et au MNA à T0. De façon intéressante, 22 métabolites étaient communs entre T0 et T7 pour la FPM ; 2 signaient simultanément les 3 critères et 15 étaient spécifiquement associés au MNA à T0. Les métabolites corrélés à la FPM à T0 et à T7 ont été identifiés comme impliqués dans le métabolisme du phosphate d'inositol (valeur de p brute = 0,044 à T0 et p = 0,055 à T7), et les 21 métabolites corrélés au MNA à T0 dans le catabolisme des acides aminés à chaîne ramifiée (AACR) (p = 0,041). Dans la cohorte BASE II, aucun métabolite mesuré par RMN n'a été associé aux critères de fragilité ou à leur évolution sur les 7 ans. A contrario, certains participant au métabolisme du phosphate-inositol ou des AACR ont été identifiés chez les hommes comme étant corrélés à la FPM et au MNA, soulignant l'importance des composantes de mobilité et de nutrition dans le syndrome de fragilité. Nous poursuivons les analyses sur d'autres paramètres caractérisant plus finement la mobilité et les aspects nutritionnels, ainsi que la capacité de la signature métabolomique à prédire l'évolution des participants vers la fragilité.

Impairment of gut function is one of the explanatory mechanisms of health status decline in elderly population. These impairments involve a decline in gut digestive physiology, metabolism and immune status, and associated to that, changes in composition and function of the microbiota it harbors. Continuous deteriorations are generally associated with the development of systemic dysregulations and ultimately pathologies that can worsen the initial health status of individuals. All these alterations observed at the gut level can then constitute a wide range of potential targets for development of nutritional strategies that can impact gut tissue or associated microbiota pattern. This can be key, in a preventive manner, to limit gut functionality decline, or in a curative way to help maintaining optimum nutrients bioavailability in a context on increased requirements, as frequently observed in pathological situations. The aim of this review is to give an overview on the alterations that can occur in the gut during aging and lead to the development of altered function in other tissues and organs, ultimately leading to the development of pathologies. Subsequently is discussed how nutritional strategies that target gut tissue and gut microbiota can help to avoid or delay the occurrence of aging-related pathologies.

Obesity is a major contributor to the silent and progressive development of type 2 diabetes (T2D) whose prevention could be improved if individuals at risk were identified earlier. Our aim is to identify early phenotypes that precede T2D in diet-induced obese minipigs. We fed four groups of minipigs (n = 5–10) either normal-fat or high-fat high-sugar diet during 2, 4, or 6 months. Morphometric features were recorded, and metabolomics and clinical parameters were assessed on fasting plasma samples. Multivariate statistical analysis on 46 morphometrical and clinical parameters allowed to differentiate 4 distinct phenotypes: NFC (control group) and three others (HF2M, HF4M, HF6M) corresponding to the different stages of the obesity progression. Compared to NFC, we observed a rapid progression of body weight and fat mass (4-, 7-, and tenfold) in obese phenotypes. Insulin resistance (IR; 2.5-fold increase of HOMA-IR) and mild dyslipidemia (1.2- and twofold increase in total cholesterol and HDL) were already present in the HF2M and remained stable in HF4M and HF6M. Plasma metabolome revealed subtle changes of 23 metabolites among the obese groups, including a progressive switch in energy metabolism from amino acids to lipids, and a transient increase in de novo lipogenesis and TCA-related metabolites in HF2M. Low anti-oxidative capacities and anti-inflammatory response metabolites were found in the HF4M, and a perturbed hexose metabolism was observed in HF6M. Overall, we show that IR and progressively obese minipigs reveal phenotype-specific metabolomic signatures for which some of the identified metabolites could be considered as potential biomarkers of early progression to TD2.

Protein turnover has been associated to residual feed intake (RFI) in beef cattle. However, this relationship may be confounded by feeding level and affected by the composition of the diet being fed. Our aim was to assess postmortem the protein metabolism signalling pathways in skeletal muscle and liver of 32 Charolais young bulls with extreme RFI phenotypes. Bulls were fed two contrasting diets during the whole fattening period but were subjected to a similar and single nutritional stimulus, induced by their respective concentrate, just prior to slaughter. The key targets were protein degradation (autophagy and ubiquitin) and synthesis signalling pathways through western-blot analysis, as well as hepatic transaminase activity. To ensure a precise assessment of all animals at the same postprandial time, they were provided with a test meal (2.5 kg of either a high-starch and high-protein concentrate or high-fibre and low-protein concentrate) 3 hours prior to slaughter, irrespective of their RFI grouping. Blood and tissues were sampled at the slaughterhouse (3 h and 3 h30 postprandially, respectively). In response to an identical single meal size, efficient RFI animals showed higher (P < 0.05) postprandial plasma β-hydroxybutyrate concentrations and insulinemia (only with the high-starch concentrate) than non-efficient animals. Moreover, efficient RFI bulls had lower muscle (P = 0.04) and liver (P = 0.08) ubiquitin protein abundance (degradation pathway) and tended to have lower alanine transaminase activity in the liver (P = 0.06) compared to non-efficient bulls, regardless of diet. A positive correlation between protein degradation potential and amino acid catabolism was identified in this study (r = 0.52, P = 0.004), which was interpreted as being biologically linked to the RFI phenotype. Efficient RFI bulls also had a faster potential for protein synthesis in the muscle, as indicated by their greater ratio of phosphorylated to total form of ribosomal protein S6 kinase (P = 0.05), regardless of diet. Results on protein synthesis pathway in muscle and plasma metabolite concentrations suggested that efficient RFI cattle may have a faster nutrient absorption and insulin responsiveness after feeding than inefficient cattle. We did not find significant differences in hepatic protein synthesis pathways between the two RFI groups (P > 0.05). Our findings suggest that, in response to an identical single meal size, efficient RFI animals exhibited lower activation of tissue protein degradation pathways and faster muscle protein synthesis activation compared to their inefficient counterparts. This pattern was observed regardless of the composition of the tested meals.

Purpose of review An increase in the plant-based characteristics of the diet is now recommended for human and planetary health. There is growing evidence that plant protein (PP) intake has beneficial effects on cardiometabolic risk. However, proteins are not consumed isolated and the protein package (lipid species, fiber, vitamins, phytochemicals, etc) may contribute, besides the protein effects per se, to explain the beneficial effects associated with PP-rich diets. Recent findings Recent studies have shown the potential of nutrimetabolomics to apprehend the complexity of both the human metabolism and the dietary habits, by providing signatures associated to the consumption of PP-rich diets. Those signatures comprised an important proportion of metabolites that were representative of the protein package, including specific amino acids (branched-chain amino acids and their derivates, glycine, lysine), but also lipid species (lysophosphatidylcholine, phosphatidylcholine, plasmalogens) and polyphenol metabolites (catechin sulfate, conjugated valerolactones and phenolic acids). Summary Further studies are needed to go deeper in the identification of all metabolites making part of the specific metabolomic signatures, associated to the large range of protein package constituents and their effects on the endogenous metabolism, rather than to the protein fraction itself. The objective is to determine the bioactive metabolites, as well as the modulated metabolic pathways and the mechanisms responsible for the observed effects on cardiometabolic health.

This study explored plasma biomarkers and metabolic pathways underlying feed efficiency measured as residual feed intake ( RFI ) in Charolais heifers. A total of 48 RFI extreme individuals (High-RFI, n = 24; Low-RFI, n = 24) were selected from a population of 142 heifers for classical plasma metabolite and hormone quantification and plasma metabolomic profiling through untargeted LC-MS. Most efficient heifers (Low-RFI) had greater (P = 0.03) plasma concentrations of IGF-1 and tended to have (P = 0.06) a lower back fat depth compared to least efficient heifers. However, no changes were noted (P ≥ 0.10) for plasma concentrations of glucose, insulin, non-esterified fatty acids, β-hydroxybutyrate and urea. The plasma metabolomic dataset comprised 3,457 ions with none significantly differing between RFI classes after false discovery rate correction (FDR > 0.10). Among the 101 ions having a raw P < 0.05 for the RFI effect, 13 were putatively annotated by using internal databases and 6 compounds were further confirmed with standards. Metabolic pathway analysis from these 6 confirmed compounds revealed that the branched chain amino acid metabolism was significantly (FDR < 0.05) impacted by the RFI classes. Our results confirmed for the first time in beef heifers previous findings obtained in male beef cattle and pointing to changes in branched-chain amino acids metabolism along with that of body composition as biological mechanisms related to RFI. Further studies are warranted to ascertain whether there is a cause-and-effect relationship between these mechanisms and RFI.

This review focuses on the added value provided by a research strategy applying metabolomics analyses to assess phenotypic flexibility in response to different nutritional challenge tests in the framework of metabolic clinical studies. We discuss findings related to the Oral Glucose Tolerance Test (OGTT) and to mixed meals with varying fat contents and food matrix complexities. Overall, the use of challenge tests combined with metabolomics revealed subtle metabolic dysregulations exacerbated during the postprandial period when comparing healthy and at cardiometabolic risk subjects. In healthy subjects, consistent postprandial metabolic shifts driven by insulin action were reported (e.g., a switch from lipid to glucose oxidation for energy fueling) with similarities between OGTT and mixed meals, especially during the first hours following meal ingestion while differences appeared in a wider timeframe. In populations with expected reduced phenotypic flexibility, often associated with increased cardiometabolic risk, a blunted response on most key postprandial pathways was reported. We also discuss the most suitable statistical tools to analyze the dynamic alterations of the postprandial metabolome while accounting for complexity in study designs and data structure. Overall, the in-depth characterization of the postprandial metabolism and associated phenotypic flexibility appears highly promising for a better understanding of the onset of cardiometabolic diseases.