In order to lower the antinutritional activity of serine protease inhibitors, we need to understand their role in seed development.

Proteases, including serine proteases, are involved in the entire life cycle of plants. Proteases are controlled by protease inhibitors (PI) to limit any uncontrolled or harmful protease activity. The role of PIs in biotic and abiotic stress tolerance is well documented, however their role in various other plant processes has not been fully elucidated. Seed development is one such area that lack detailed work on the function of PIs despite the fact that this is a key process in the life cycle of the plant. Serine protease inhibitors (SPI) such as the Bowman-Birk inhibitors and Kunitz-type inhibitors, are abundant in legume seeds and act as antinutrients in humans and animals. Their role in seed development is not fully understood and present an interesting research target. Whether lowering the levels and activity of PIs, in order to lower the anti-nutrient levels in seed will affect the development of viable seed, remains an important question. Studies on the function of SPI in seed development are therefore required. In this Perspective paper, we provide an overview on the current knowledge of seed storage proteins, their degradation as well as on the serine protease-SPI system in seeds and what is known about the consequences when this system is modified. We discuss areas that require investigation. This includes the identification of seed specific SPIs; screening of germplasms, to identify plants with low seed inhibitor content, establishing serine protease-SPI ratios and lastly a focus on molecular techniques that can be used to modify seed SPI activity.

Relative validity of the Planetary Health Diet Index by comparison with usual nutrient intakes, plasma food consumption biomarkers, and adherence to the Mediterranean diet among European adolescents: the HELENA study.

PURPOSE: The EAT-Lancet Commission proposed an evidence-based global reference diet to improve human health within planetary boundaries. Recently, the Planetary Health Diet Index (PHDI) was developed based on the EAT-Lancet recommendations and validated among Brazilian adults. However, the relative validity of the PHDI in adolescents has yet to be assessed. Thus, we aimed to evaluate the relative validity of the PHDI in European adolescents. METHODS: We used cross-sectional data from 1804 adolescents (12.5-17.5 years) enrolled in the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study. The PHDI (0-150 points) was calculated based on dietary intake data from two non-consecutive 24-h dietary recalls. Associations between the PHDI and usual nutrient intakes, plasma food consumption biomarkers, and adherence to the Mediterranean diet were evaluated using multivariable-adjusted mixed-effects linear regression models. RESULTS: Higher PHDI score was associated with greater intakes of nutrients predominantly from plant-source foods, such as vegetable protein, vitamin E, and folate and with lower intake of nutrients predominately from animal-source foods, such as total and saturated fat, cholesterol, and animal protein. Furthermore, a higher PHDI score was also positively associated with plasma ß-carotene, vitamin C, vitamin D, folate, and ferritin concentrations, while negatively associated with trans-fatty acids concentration. Moreover, higher PHDI was related to a greater adherence to the Mediterranean dietary pattern. CONCLUSIONS: The PHDI showed good relative validity among adolescents in the HELENA study. Hence, future research should assess adherence to the PHDI and long-term health outcomes.

Changes in physical activity patterns from adolescence to young adulthood: the BELINDA study.

Physical activity (PA) is recognized as a marker of health. The aim was to investigate PA differences from adolescence to young adulthood. European adolescents included in the HELENA study were invited to participate in a follow-up study, 10 years later. The present study included 141 adults (25.0 ± 1.4 years) for whom valid accelerometer data were available in adolescence and adulthood. Changes in PA by sex, weight and maternal education level were explored with interactions. Time spent in sedentary activity, light PA (LPA) and moderate PA (MPA) increased by 39.1, 59.6 and 6.6 min/day, respectively, whereas the time spent in vigorous PA (VPA) decreased by 11.3 min/day compared with adolescent VPA (p < 0.05). Increases in MPA were greater on weekends compared with weekdays, but we found a greater decrease in VPA on weekdays compared with weekends. Moderate-to-vigorous PA (MVPA) decreased significantly on weekdays (-9.6 min/day; 95%CI, -15.9 to -3.4), while it increased on weekends (8.4 min/day; 95%CI, 1.9 to 14.8). Significant heterogeneity was found across sexes for VPA and MVPA, with a stronger decrease in VPA in males compared with females and a significant decrease in MVPA (-12.5 min/day; 95%CI, -20.4 to -4.5) in males but not in females (1.9 min/day; 95%CI, -5.5 to 9.2). No significant heterogeneity was found to be linked to maternal education level or weight, irrespective of PA level.  Conclusion: Our data suggest that the transition from adolescence to young adulthood is a critical period for lifestyle PA habits. A decline in VPA and an increasingly sedentary time were observed. The observed changes are worrying and may increase the risk of developing adverse health consequences later in life. What is Known: • The transition from adolescence to adulthood is marked by many life changes affecting lifestyle habits. Most studies tracking physical activity from adolescence to adulthood were done using PA questionnaires, which is a subjective method. What is New: • Our study bring first data on objective changes in PA patterns between adolescence and young adulthood, taking account of BMI, sex and maternal educational level. Our results suggest that the transition from adolescence to young adulthood is a critical period for lifestyle PA habits, especially for time spent on sedentary activities.

The Alignment of Recommendations of Dietary Guidelines with Sustainability Aspects: Lessons Learned from Italy's Example and Proposals for Future Development.

The main objective of this paper is to describe the process of the inclusion of sustainability in the Italian Dietary Guidelines (IDGs). In the IDGs' sustainability chapter, particular emphasis was put on the selection of foods, recommending a plant-based diet with a large quota of vegetable proteins. Advice was also given on the selection of local seasonal products, with low growth input, such as fertilizers, artificial light and heating. Reduction of animal food was recommended, to be substituted with lower impact foods (poultry, milk, eggs and Mediterranean fish including aquaculture). Food waste was largely addressed. Recommendations were made for food purchase planning, food storage and the reuse of leftovers as strategies to reduce waste and save money. The IDGs sustainability recommendations were related to the 16 guiding principles of a sustainable healthy diet and their contribution to the achievement of the Sustainable Developing Goals was provided. The inclusion of sustainability in dietary guidelines requires a multidisciplinary approach to cover the wide range of aspects of a sustainable diet. In the IDGs, it was possible to show that practical recommendations for improving sustainability behavior can be passed on to consumers. Methodological aspects for developing recommendations are not definitive.

Dietary assessment training: The Italian IV SCAI study on 10-74 year-old individuals' food consumption.

Dietary surveys are conducted to examine the population's dietary patterns that require a complex system of databases, and rules for constructing the data matrix (precision, coding, deriving new variables, e.g., body mass index from individual's height and weight, classes, e.g., age-class, socio-economic status, physical activity, etc.). Management of the data collection requires specialized fieldworkers to allow for the collection of harmonized and standardized data. In this way, only statistical variability is envisaged and any eventual biases are due to probabilistic distribution but data are not affected by inaccuracy. Training the fieldworkers is a crucial part of each dietary survey. The idea to provide constant training throughout the whole survey period, from the preparatory phase to the data collection phase, relies on the necessity to train fieldworkers and monitor the skills acquired during the study, in addition to helping fieldworkers to gain the necessary experience. This study aims to relate the experience in conducting the course path to high specialized interviewers who carried out the cycle devoted to the 10-74 age class of the fourth nationwide food consumption study in Italy (IV SCAI ADULT) according to the European Food Safety Authority (EFSA) guide. A course path was structured in three steps corresponding to the preparation, pilot, and collection phases. The whole path achieved the goal of collecting data related to 12 individuals by each participant, with an overall success rate (successful trainees/total participants) of 16.8% (84 out of an initial 500). The study aimed to provide good quality data in the short term and a highly specialized community in the long term. Surveillance nutritional systems can count on a highly skilled community, so decision-making in public health nutrition and a sustainable and healthy food system can rely on this infrastructure.

Antinutritional factors, nutritional improvement, and future food use of common beans: A perspective.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean's nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

The carbon footprint of Italian schools meals: An optimal choice of dishes in vegan, vegetarian, and omnivorous menus.

This study aims to assess the carbon footprint associated with vegan, vegetarian, and omnivorous menus for primary school lunches in Italy. For this purpose, healthy and acceptable menus with minimal greenhouse gas emissions have been designed by a binary linear programming model. The results show that the adoption of a specific diet may help in reducing the carbon footprint of menus, but it is the optimal selection of dishes that ultimately makes the difference. Interestingly enough, the optimal choice of dishes and the restriction of meat consumption in omnivorous menus can lead up to a 40% emission reduction compared to the current school lunch menu of the municipality of Rome. Moreover, the optimal choice of dishes in vegan menus provides the menu with the lowest carbon footprint among all kinds of diets.

Identification of Lifestyle Risk Factors in Adolescence Influencing Cardiovascular Health in Young Adults: The BELINDA Study.

Cardiovascular diseases are the leading cause of mortality worldwide. These diseases originate in childhood, and a better understanding of their early determinants and risk factors would allow better prevention. The BELINDA (BEtter LIfe by Nutrition During Adulthood) study is a 10−14-year follow-up of the HEalthy Lifestyle in Europe by Nutrition in Adolescence study (the HELENA study, a European cross-sectional study in adolescents). The study aims to evaluate cardiovascular risk using the PDAY (Pathobiological Determinants of Atherosclerosis in Youth) risk score during young adulthood (21−32 years), and to examine the impact of risk factors identified during adolescence (12.5−17.5 years). Our secondary objective is to compare the characteristics of the BELINDA study population with the HELENA population not participating in the follow-up study. The HELENA study recruited 3528 adolescents during 2006−2007 and reassessed 232 of them 10−14 years later as young adults. We assessed clinical status, anthropometry, nutrition, physical activity (including sedentary behavior), physical fitness, and mental health parameters, and collected biological samples (blood, stool, and hair). Dietary intake, and physical activity and fitness data were also collected. A multivariable linear regression model will be used for the analysis of the primary outcome. A Chi-square and T-test were conducted for the comparison of the descriptive data (gender, age, weight, height, body mass index (BMI), and maternal school level) between participating and non-participating BELINDA adolescents. When comparing the 1327 eligible subjects with the 232 included in the BELINDA study, no significant differences regarding gender (p = 0.72), age (p = 0.60), height (p = 0.11), and weight (p = 0.083) at adolescence were found. However, the participating population had a lower BMI (20.4 ± 3.1 kg/m2 versus 21.2 ± 3.6 kg/m2; p < 0.001) and a higher maternal educational level (46.8% high school or university level versus 38.6%; p = 0.027) than the HELENA population who did not participate in the BELINDA study. The complete phenotyping obtained at adolescence through the HELENA study is a unique opportunity to identify adolescent risk factors for cardiovascular diseases. This paper will serve as a methodological basis for future analysis of this study.

Does Plant Breeding for Antioxidant-Rich Foods Have an Impact on Human Health?

Given the general beneficial effects of antioxidants-rich foods on human health and disease prevention, there is a continuous interest in plant secondary metabolites conferring attractive colors to fruits and grains and responsible, together with others, for nutraceutical properties. Cereals and Solanaceae are important components of the human diet, thus, they are the main targets for functional food development by exploitation of genetic resources and metabolic engineering. In this review, we focus on the impact of antioxidants-rich cereal and Solanaceae derived foods on human health by analyzing natural biodiversity and biotechnological strategies aiming at increasing the antioxidant level of grains and fruits, the impact of agronomic practices and food processing on antioxidant properties combined with a focus on the current state of pre-clinical and clinical studies. Despite the strong evidence in in vitro and animal studies supporting the beneficial effects of antioxidants-rich diets in preventing diseases, clinical studies are still not sufficient to prove the impact of antioxidant rich cereal and Solanaceae derived foods on human.

Corrigendum: A Dietary Assessment Training Course Path: The Italian IV SCAI Study on Children Food Consumption.

[This corrects the article DOI: 10.3389/fpubh.2021.590315.].

The Promotions of Sustainable Lunch Meals in School Feeding Programs: The Case of Italy.

School is considered a privileged environment for health education and school feeding represents an opportunity for promoting sustainable foods to young generations. The objective of this paper is to demonstrate that is possible to select, from existing school menus, recipes that combine healthy foods with low environmental impact. A national sample of Italian school menus was collected and a total number of 194 recipes were included on a database containing 70 first courses, 83 s courses, 39 side dishes, 1 portion of fruit, and 1 portion of bread. A mathematical model was conceived to combine nutritional adequacy and acceptability criteria while minimizing GHGs emissions. The result is a four-week menu characterized by large vegetable components that were used not only as side dishes but also as ingredients in the first and second courses. Legumes and pasta are often included, and white meat is selected instead of red meat. The findings presented in this paper demonstrated that it is possible to design environmental-friendly meals from existing school menus. The mathematical model developed in this work has the potentiality of being completely scalable, easily updatable, and widely utilizable in different settings either for design or monitoring purposes as well as for research data collection.

A Dietary Assessment Training Course Path: The Italian IV SCAI Study on Children Food Consumption.

The eating patterns in a population can be estimated through dietary surveys in which open-ended assessment methods, such as diaries and interviews, or semi-quantitative food frequency questionnaires are administered. A harmonized dietary survey methodology, together with a standardized operational procedure, in conducting the study is crucial to ensure the comparability of the results and the accuracy of information, thus reducing uncertainty and increasing the reliability of the results. Dietary patterns (i) include several target variables (foods, energy and nutrients, other food components), (ii) require several explanatory variables (age, gender, anthropometric measurements, socio-cultural and economic characteristics, lifestyle, preferences, attitudes, beliefs, organization of food-related activities, etc.), and (iii) have impacts in several domains: imbalance diets; acute and chronic exposures affect health, specifically non-communicable diseases; and then sanitary expenditure. On the other hand, food demand has impacts on the food system: production, distribution, and food services system; food wastes and other wastes generated by food-related activities of the households (e.g., packaging disposal) have consequences on the "health of the planet" which in turn can have effects on human health. Harmonization and standardization of measurement methods and procedures in such a complex context require an ad hoc structured information system made by databases (food nomenclatures, portion sizes, food atlas, recipes) and methodological tools (quantification methods, food coding systems, assessment of nutritional status, data processing to extrapolate what we consider validated dietary data). Establishing a community of professionals specialized in dietary data management could lead to build a surveillance system for monitoring eating habits in the short term, thus reducing costs, and to arrange a training re-training system. Creating and maintaining the dietary data managers community is challenging but possible. In this context, the cooperation between the CREA Research Centre for Food and Nutrition and the Italian National Health Institute (ISS) promoted and supported by the Italian Ministry of Health may represent a model of best practice that can ensure a continuous training for the professional community carrying out a nutritional study.

Could Dietary Goals and Climate Change Mitigation Be Achieved Through Optimized Diet? The Experience of Modeling the National Food Consumption Data in Italy.

Objective: The aim of this study is to define a healthy and sustainable diet model with low GHGE, fulfilling dietary requirements, and considering current Italian food consumption patterns. Design: A duly designed database was developed, linking food nutritional composition and GHGE based on 921 food items consumed in Italy according to the last national food consumption survey (INRAN-SCAI 2005-2006). Linear programming was used to develop new diet plans separately for males and females, aged 18-60 years (n = 2,098 subjects), in order to minimize GHGE. The program is based on dietary goals and acceptability constraints as well as on 13 nutrient requirement constraints aiming to reach a healthy and acceptable diet for the Italian population. Results: Diet optimization resulted in a nutritionally adequate pattern minimizing GHGE values (4.0 vs. 1.9 kg CO2e/day for males and 3.2 vs. 1.6 kg CO2e/day for females). In both sexes, the nutrient intake of the optimized diet was at the established lower bound for cholesterol and calcium and at the established upper bound for free sugar and fiber. In males, intake of zinc was at the established lower bound whereas iron was at the established upper bound. Consumption of red meat and fruit and vegetables was at the established lower and upper bound, respectively, in both males and females. Despite the decrease in meat consumption, especially red meat, in the optimized diet with respect to the observed diet, levels of iron intake in females increased by 10% (10.3 vs. 11.3 mg/day) but remained below the adequate intake established in Italian national DRIs. Conclusions: An attainable healthy dietary pattern was developed that would lead to the reduction of GHGE by 48% for males and by 50% for females with respect to current food consumption in the Italian adult population. Health-promoting dietary patterns can substantially contribute to achieve related Sustainable Development Goals.

Total Polyphenol Intake Is Inversely Associated with a Pro/Anti-Inflammatory Biomarker Ratio in European Adolescents of the HELENA Study.

BACKGROUND: Although high dietary polyphenol intake is negatively associated with risk of certain inflammation-associated chronic diseases, the underlying mechanisms are not fully understood and few studies have explored this in adolescents. OBJECTIVE: This study aimed to evaluate the association between intakes of total polyphenols, polyphenol classes, and the 10 most commonly consumed individual polyphenols with inflammatory biomarkers in the blood of European adolescents. METHODS: In the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) Study, 526 adolescents (54% girls; 12.5-17.5 y) had data on inflammatory biomarkers and polyphenol intake from 2 nonconsecutive 24-h recalls via matching with the Phenol-Explorer database. Inflammatory biomarkers in serum were IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, transforming growth factor ß1 (TGF-ß1), TNF-α, IFN-γ, soluble vascular adhesion molecule 1 (sVCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble E-selectin (sE-selectin), white blood cells, lymphocytes, T cells, and C-reactive protein. Multilevel linear models were used to test associations of polyphenol intake with a pro/anti-inflammatory biomarker ratio [(zTNF-α + zIL-6 + zIL-1)/3/zIL-10] as well as with separate inflammatory biomarkers, adjusted for sociodemographic variables, diet inflammation index, BMI z score, and serum triglycerides. RESULTS: The pro/anti-inflammatory biomarker ratio was linearly inversely associated with the intake of total polyphenols (ß = -0.11, P = 0.040). When other inflammation biomarkers were considered, the serum IL-10 concentration was inversely associated with total polyphenol (ß = -0.12, P = 0.017) and flavonoid (ß = -0.12, P = 0.013) intakes, findings that were inconsistent with the biomarker ratio results. However, the anti-inflammatory capacity of polyphenols was confirmed by positive associations of IL-4 with phenolic acid (ß = 0.09 P = 0.049) and stilbene (ß = 0.13, P = 0.019) intakes and the negative association of IL-1, IL-2, and IFN-γ with lignan intake (ß = -0.10, P = 0.034; ß = -0.09, P = 0.049; ß = -0.11, P = 0.023). CONCLUSIONS: The negative relation with the overall pro/anti-inflammatory biomarker ratio suggests a potential anti-inflammatory role of high polyphenol intakes among European adolescents. Nevertheless, associations are dependent on polyphenol type and the inflammatory biomarker measured.

Interplay between the Mediterranean diet and C-reactive protein genetic polymorphisms towards inflammation in adolescents.

AIM: From a nutrigenetics perspective, we aim to investigate the moderating role of the Mediterranean diet and each of its subgroups in the association between C-reactive protein (CRP) gene polymorphisms and CRP blood concentration in adolescents. METHODS: In 562 adolescents (13-17 y) of the European HELENA study, data was available on circulating CRP levels as inflammatory biomarker, three CRP gene SNPs (rs3093068, rs1204, rs1130864), food intake determined by a self-administered computerized 24 h-dietary recall for 2 days, and body composition. A 9-point Mediterranean diet score and each food subgroup were tested as moderator via SNP*diet interaction. Analyzes were adjusted for age, sex, puberty, adiposity and socioeconomic status. RESULTS: The minor allele frequencies of rs3093068 and rs1130864 SNPs (GG and TT, respectively) were associated with higher CRP concentrations, while rs1205 (CT/TT) was associated with lower CRP concentrations. There were significant interactions between rs3093068 and Mediterranean diet (B = -0.1139, p = 0.011), or the fish food subgroup (B = -0.0090, p = 0.022), so that those with the highest genetic CRP risk underwent the highest CRP attenuation by a healthier diet. Although the effect of diet and SNP was substantial, the explained variance by interaction was only 1%. CONCLUSION: Greater adherence to the Mediterranean diet and particularly its fish component was associated with a lower CRP blood concentrations especially in those at highest genetic risk due to the rs3093068 SNP.

Diet as a moderator in the association of sedentary behaviors with inflammatory biomarkers among adolescents in the HELENA study.

AIM: To assess if a healthy diet might attenuate the positive sedentary-inflammation relation, whereas an unhealthy diet may increase the effect of sedentary behaviors on inflammatory biomarkers. METHODS: In 618 adolescents (13-17 years) of the European HELENA study, data were available on body composition, a set of inflammation markers, and food intake assessed by a self-administered computerized 24 h dietary recall for 2 days. A 9-point Mediterranean diet score and an antioxidant-rich diet z-score were used as dietary indices and tested as moderators. A set of low-grade inflammatory characteristics was used as outcome: several cytokines in an inflammatory ratio (IL-6, IL-10, TNF-α, TGFß-1), C-reactive protein, three cell-adhesion molecules (sVCAM-1, sICAM-1, sE-selectin), three cardiovascular risk markers (GGT, ALT, homocysteine) and three immune cell types (white blood cells, lymphocytes, CD3). Sedentary behaviors were self-reported and analyzed as total screen time. Multiple linear regression analyses tested moderation by diet in the sedentary behaviors-inflammation association adjusted for age, sex, country, adiposity (sum of six skinfolds), parental education, and socio-economic status. RESULTS: Both diet scores, Mediterranean and antioxidant-rich diet, were significant protective moderators in the effect of sedentary behaviors on alanine-transaminase enzyme (P = 0.014; P = 0.027), and on the pro/anti-inflammatory cytokine ratio (P = 0.001; P = 0.004), but not on other inflammatory parameters. CONCLUSION: A higher adherence to the Mediterranean diet or an antioxidant-rich diet may attenuate the onset of oxidative stress signs associated by sedentary behaviors, whereas a poor diet seems to increase inflammation.

Diet as moderator in the association of adiposity with inflammatory biomarkers among adolescents in the HELENA study.

AIM: Our aim is to demonstrate that a healthy diet might reduce the relation between adiposity and inflammation, whereas an unhealthy diet may increase the effect of adiposity on inflammatory biomarkers. METHODS: In 618 adolescents (13-17 years) of the European HELENA study, data were available on body composition, a set of inflammation markers, and food intake determined by a self-administered computerized 24-h recall. A 9-point Mediterranean diet score and an antioxidant-rich diet score were used as dietary parameters and tested as moderator. Total body fat was represented by the sum of six skinfold thicknesses and central adiposity by waist circumference. A set of inflammation-related biomarkers was used as outcome: a pro/anti-inflammatory interleukins ratio, TGFß-1, C-reactive protein, TNF-α, 3 cell adhesion molecules, and 3 types of immune cells; gamma-glutamyltransferase (GGT) and homocysteine were used as cardiovascular disease risk biomarkers, and alanine transaminase (ALT) as liver dysfunction biomarker. Multiple linear regression analyses tested moderation by diet in the adiposity-inflammation association and were adjusted for age, sex, country, puberty, socioeconomic status. RESULTS: Both the Mediterranean and antioxidant-rich diet, and overall and central adiposity, were important in the moderation. Diet was a significant protective moderator in the effect of adiposity on the pro/anti-inflammatory interleukins ratio, TGFß-1, GGT, and ALT. CONCLUSION: In conclusion, in some cases, a diet rich in antioxidants and essential nutrients may attenuate the concentration of inflammatory biomarkers caused by adiposity, whereas a poor diet appears to contribute to the onset of early oxidative stress signs.

Lifestyle patterns and endocrine, metabolic, and immunological biomarkers in European adolescents: The HELENA study.

OBJECTIVE: To evaluate the association of lifestyle patterns related to physical activity (PA), sedentariness, and sleep with endocrine, metabolic, and immunological health biomarkers in European adolescents. METHODS: The present cross-sectional study comprised 3528 adolescents (1845 girls) (12.5-17.5 years) enrolled in the Healthy Lifestyle in Europe by Nutrition in Adolescence Study. Cluster analysis was performed by including body composition, PA by accelerometry, self-reported sedentary behaviors, and sleep duration. We also measured endocrine, metabolic, and immunological biomarkers. RESULTS: Three-cluster solutions were identified: (a) light-PA time, moderate-vigorous-PA time and sedentary time, (b) light-PA time, moderate-vigorous-PA time, sedentary time and sleep time, (c) light-PA time, moderate-vigorous-PA time, sedentary time and body composition. In addition, each cluster solution was defined as: "healthy," "medium healthy," and "unhealthy" according to the presented rating. Analysis of variance showed that overall the healthiest groups from the three clusters analyzed presented a better metabolic profile. A decision tree analysis showed that leptin had a strong association with cluster 3 in both boys and girls, high-density lipoprotein cholesterol had the strongest association with clusters 1 and 3 in boys. Cortisol had the strongest association with cluster 1. HOMA index (homeostatic model assessment) and C3 showed a strong association with cluster 3 in girls. CONCLUSIONS: Our results support the existence of different interactions between metabolic health and lifestyle patterns related to PA, sedentariness, and sleep, with some gender-specific findings. These results highlight the importance to consider multiple lifestyle-related health factors in the assessment of adolescents' health to plan favorable strategies.