How exercise, physical activity and diet modulate immune and stress responses

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H

OW EXERCISE

,

PHYSICAL ACTIVITY AND DIET

MODULATE IMMUNE AND STRESS RESPONSES

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INAL VERSION

Diana Margarida Gonçalves Solha Pereira da Silva

Doctoral Program in Clinical Investigation

and Health Services Research

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Supervisor:

André Moreira, MD Ph.D.

Invited associate Professor of Immunology, Faculty of Medicine of Porto University Medical Consultant of Immunoallergology, Centro Hospitalar São João, Porto

Co-supervisor

Joana Carvalho, Ph. D.

Associate Professor in Sports Faculty of Porto University

Institutions:

Faculty of Medicine, University of Porto Alameda Prof. Hernâni Monteiro

4200 – 319 Porto, Portugal

Centro Hospitalar de São João, EPE Alameda Prof. Hernâni Monteiro 4200 – 319 Porto, Portugal

Faculty of Sports of University of Porto Rua Dr. Plácido Costa, 91

4200 – 450 Porto, Portugal

Faculty of Nutrition and Food Sciences, University of Porto Rua Dr. Roberto Frias

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Elements of the Jury:

Professor Rui Manuel Lopes Nunes, cathedratic professor of the Faculty of Medicine of Porto University (President)

Professor Tari Markku Kallevi Haatela, emeritus professor of the Helsinki University Professor Vanessa Garcia Larsen, assistant professor of the Johns Hopkins

Bloomberg School of Public Health, Baltimore, United States

Professor Sílvia Maria da Rocha Simões Carriço, assistant professor of the Aveiro University

Professor José Luís Dias Delgado, associated professor with aggregation of the Faculty of Medicine of Porto University

Professor André Miguel Afonso Sousa Moreira, invited associated professor with aggregation of the Faculty of Medicine of Porto University (Supervisor)

Professor Pedro Alexandre Afonso de Sousa Moreira, cathedratic professor of the Faculty of Nutrition and Food Sciences of Porto University

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Ao Manuel e à Francisca Aos meus pais Ao Alberto

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“No texto está o tempo: palavras escritas no passado, lidas no presente, e o entendimento, suspeita fulgurante, ambição um tanto pretensiosa, exatamente no lugar onde imaginamos o futuro” José Luís Peixoto

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List of original publications

The present thesis is based on the following publications, which will be referred in the manuscript by their roman numerals:

I- Meal-Exercise Challenge and Physical Activity Reduction Impact on Immunity and Inflammation (MERIIT trial)- Contemp Clin Trials Commun. 2018 9; 10:177-189.doi: 10.1016/j.conctc.2018.05.010.

II- Immune response to a meal-exercise challenge and short-term reduction of physical activity III- Acute exercise and physical activity impact on the allostatic load: evidence from two steps clinical trial (submitted to Scandinavian journal of medicine & science in sports)

IV- What is the effect of a Fast Food versus a Mediterranean meal in the adipokine response to an exercise challenge? PLoS ONE 14(4):e0215475.https://doi.org/

10.1371/journal.pone.0215475

V- Impact of long-term exercise training on the metabolomic profile and respiratory infections risk in older adults Eur J Sport Sci. 2019;19(3):384-393. doi:10.1080/17461391.2018.1499809 VI- Respiratory infection in elite swimmers – a prospective surveillance study

VII- A Mediterranean compared with fast-food type meal improves autonomic nervous system response

VIII- Effects of weight changes in the autonomic nervous system: A systematic review and meta-analysis Clin Nutr. 2019;38(1):110-126.doi: 10.1016/j.clnu.2018.01.006

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Abbreviations

ACV - Average constriction velocity ADV - Average dilation velocity ANS - Autonomic nervous system BRS - Baroreflex sensitivity BMI - Body mass index CCT - Controlled clinical trial

CDC/NHSN – Centers for Disease Control and Prevention/National healthcare safety network CI - confidence interval

CON - percentage of pupil constriction eNO - exhaled nitric oxide

GRADE - Grading of Recommendations Assessment, Development, and Evaluation FEF25-75 - Forced expiratory flow at 25-75% of vital capacity

FEV1 - Forced expiratory volume at the first second FVC - Forced vital capacity

GCxGC-ToF-MS - Double GC coupled to a time-of-flight mass-spectrometer HF - High Frequency

HFnu - High Frequency in normalized units HRR – Heart rate recovery

HRV – Heart rate variability IL- Inter-leukine

LF – Low frequency

LF/HF ratio – Low Frequency/ High Frequency ratio LFnu – Low Frequency in normalized units

MCV - Maximum conscription velocity MSNA – Muscle sympathetic nerve activity NA-SR - Noradrenaline spillover rate NK- natural killer cells

OR - Odds ratio

PD20- Provocative dose causing a 22% fall in forced expiratory volume in one second PRISMA - Preferred Reporting Items for Systematic Reviews and Meta-Analyses RI- Respiratory Infection

RMSSD - Root mean square of successive differences SD - Standard deviation

SD1 - Standard deviation of instantaneous beat-to-beat R-R interval variability

SD1nu - Standard deviation of instantaneous beat-to-beat R-R interval variability normalized SDNN – Standard deviation of normal-to-normal intervals

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17 Th- T helper cell

TP - Total Power

T75 - Time in seconds at 75% recovery of pupil size VLF - Very low frequency

VOC/VOCs - Volatile organic compound/s WHO- world health organization

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Abstract

Exercise, sedentary behavior and dietary patterns are important lifestyle factors that determine the risk of noncommunicable chronic diseases. Metabolic and immune systems linked in the “immunometabolism” concept mediate the response to stress and lifestyle changes. An acute exercise bout, acute reduction of physical activity or long-term exercise induce different responses. They are dependent on the individual exposome, that is controlled by linear incremental exposure, like age and specific external exposures, namely occupational ones, as in swimmers. These aspects are closely linked with the autonomic nervous system as it controls acute and long-term responses to stress. Diet can change this relationship, in a positive or negative direction, both modulating immune, metabolic, allostatic and autonomic responses.

Few studies have addressed the acute impact of exercise or of a meal on the modulation of these responses, and there is also limited evidence how exercise links to metabolic-oxidative stress related parameters in the modulation of the susceptibility to infections.

The aim of this thesis was to investigate the effects of exercise, physical activity and diet on the immune, metabolic and stress responses.

This thesis is based on three study types: a clinical trial; a prospective surveillance study and a systematic review and meta-analysis. Clinical trial designs were applied on: a) a two steps clinical trial including a randomized crossover clinical trial, comparing the effect of a fast-food type of meal, with an isoenergetic similar Mediterranean type of meal in the immune response to an exercise challenge and a pilot trial assessing the neuro-immune-endocrine change induced by acute decreasing by half the usual physical activity level; b) a controlled clinical trial evaluating the impact of 8 months exercise training on the risk of respiratory infections and on the metabolomic profile of oxidative stress lipid peroxidation-related metabolites in older adults. The prospective 14 weeks` surveillance study evaluated the risk of respiratory infections in high competition level swimmers during wintertime. The systematic review and meta-analysis addressed the impact of weight changes, through diet and/or exercise-based interventions, in the autonomic nervous system.

A Mediterranean type meal, compared to a fast food type meal, was associated with a lower leukocyte absolute number increase, a milder reduction of relative percentage of circulating CD19+ and CD3+ cells and a decreased lymphocyte Foxp 3 expression. Allostatic state was modulated by an acute meal and by exercise challenge, and exercise was also linked with an increase of circulatory levels of adipsin, resistin, lipocalin and PAI-1, independent of the previous meal type. A pre-exercise Mediterranean type meal potentiated the increase of adipsin

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19 after the exercise test. A fast food type meal was associated with dyslipidemia and insulin resistance related biomarkers.

A short term induced reduction of physical activity in healthy young individuals modulated immune response and metabolic response with an increase of circulating lymphocyte and natural killer cells number, a decrease in the relative percentage of CD3+ and CD19+ cells; and an increase of body fat and insulin levels with no impact on the allostatic load indexes.

Moderate long-term exercise training in older adults did not increase the risk of respiratory infections. A pattern of metabolites involved in lipid peroxidation and oxidative stress correlating with the number of respiratory infections was identified. The incidence of respiratory infections in elite swimmers was high and occurred mainly during and immediately after periods of higher intensity training.

The autonomic nervous system response to a meal was modulated by a Mediterranean meal, which counteracted the sympathetic nervous system activation and reduction of parasympathetic parameters. Diet and exercise-based weight loss appeared to increase parasympathetic activity and decrease sympathetic nerve activity.

In conclusion, we have showed in our study that a Mediterranean type of meal protects from immune, inflammatory and metabolic response to an exercise challenge. Although further studies need to address these effects on specific populations, our results could guide the most adequate pre-exercise meal for individuals engaging acute exercise activities. Our study supports that acute sedentary behaviour in healthy individuals leads to an immune and metabolic dysregulation, despite there were no changes in the allostatic load index; further studies with larger population might lead to different results. The autonomic nervous system was modulated by weight changes with a cumulative effect of diet and exercise, although even a single meal influences the autonomic nervous system response. Focusing on a larger population would allow a better understanding of the possible impact of autonomic nervous system changes in metabolic dysfunction.

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Resumo

O risco de desenvolver doenças não transmissíveis está globalmente associado ao estilo de vida, na qual se inclui a prática de exercício físico, o sedentarismo e a dieta. A reação ao stress e a adaptação a mudanças no estilo de vida depende da resposta conjunta do sistema imune e metabólico tendo como base o conceito de “imunometabolismo”.

O estímulo agudo de exercício físico, a redução da atividade física ou a adaptação crónica à prática de exercício de longo prazo traduz-se em diferentes respostas imunológicas e metabólicas. Estas, estão dependentes do expossoma de cada indivíduo. O expossoma depende da exposição linear e progressiva ao longo da vida, sendo a idade um fator importante, mas também existem outros fatores externos mais específicos, como por exemplo a exposição induzida pelas características do treino dos nadadores. O sistema nervoso autónomo atua como elo de ligação nas respostas ao stress, tanto a curto e a longo prazo. A dieta altera a direção desta relação de forma positiva ou negativa, modulando tanto a resposta imune, metabólica, alostática como autonómica.

A evidência que aborda o impacto agudo do exercício ou de uma refeição na modelação desta resposta é escassa, sendo também diminuta a evidência relativa à relação entre o exercício e a alteração de parâmetros relacionados com o stress oxidativo, o metabolismo e a suscetibilidade às infeções.

O objetivo desta tese é avaliar o efeito do exercício, da redução da atividade física e da dieta na resposta imunológica, metabólica e ao stress.

Para o objetivo exposto esta tese baseou-se em três tipos de estudos: ensaio clínico; estudo observacional prospetivo e uma revisão sistemática e meta-análise.

Foram aplicados os seguintes ensaios clínicos: a) ensaio clínico do tipo cross-over que comparou o efeito de uma refeição mediterrânica com uma refeição isoenergética do tipo fast

food na resposta imunológica e metabólica a um estímulo agudo de exercício físico e um

ensaio piloto que avaliou as alterações neuro-imuno-endócrinas da redução, durante duas semanas, do nível da atividade física para metade; b) ensaio clínico controlado que avaliou o impacto de um programa de 8 meses de exercício físico no risco de infeções respiratórias e no perfil de metabolitos de peroxidação lipídica em idosos.

No estudo prospetivo foi avaliada o número de infeções respiratórias em nadadores de alta competição durante um período de 14 semanas durante o Inverno. Na revisão sistemática e meta-análise foi estudado o impacto da alteração do peso, causada pela dieta ou exercício físico, no sistema nervoso autónomo.

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21 Os resultados mostraram que: i) uma refeição mediterrânica associou-se a um menor aumento do número de leucócitos e a uma menor redução da percentagem relativa de CD19+ e CD3 + e da expressão de Foxp 3 em linfócitos circulantes comparativamente com uma do tipo fast food; ii) independentemente do tipo de refeição prévia, o estimulo agudo de exercício físico modelou a resposta alostática e associou-se a um aumento da adipsina, resistina, lipocalina e PAI-1; por um lado, a ingestão de uma refeição mediterrânea antes do estímulo agudo de exercício associou-se a um maior aumento da adipsina em comparação com uma refeição fast food, por outro lado a refeição de tipo fast-food antes de uma prova de exercício induziu um maior aumento dos biomarcadores associados à insulino-resistência e dislipidemia; iii) uma redução de duas semanas na atividade física levou a um aumento do número de linfócitos e da percentagem relativa de células NK em circulação bem como a um declínio de percentagem relativa de linfócitos CD3+ e CD19+; paralelamente observou-se um aumento da percentagem de massa gorda e da insulina sérica, não alterando, contudo, o índice de carga alostática.; iv) um programa de exercício moderado e de longo prazo efetuado por idosos não aumentou o risco de infeção respiratória; foi possível associar o número de infeções respiratórias a um padrão de metabolitos relacionados com a peroxidação lipídica ; v) A incidência de infeções respiratórias em atletas nadadores de alta competição foi elevada e ocorreu maioritariamente durante e após períodos de maior intensidade de treino; vi) A resposta do sistema nervosa autónomo a uma refeição foi modelada pelo tipo de refeição sendo que a refeição mediterrânica contrariou a ativação do sistema nervoso simpático e a redução dos parâmetros parassimpáticos; viii) A perda de peso por dieta ou baseada na prática de exercício parece associar-se a uma hegemonia do sistema nervoso parassimpático e redução da atividade do sistema nervoso simpático.

Em conclusão, esta tese sugere que uma refeição pré-exercício do tipo mediterrânica parece proteger da resposta aguda imunológica, inflamatória e metabólica ao exercício físico. Contudo, são necessários mais estudos para avaliar a reprodutibilidade destas observações em particular em populações específicas. Estes resultados poderão guiar a escolha na refeição mais adequada a efetuar antes da prática de exercício. Por outro lado, este estudo sugere que o sedentarismo promove uma desregulação metabólica e imunológica, apesar de não se terem observado alterações na carga alostática. As alterações no peso, em particular a sua redução pela dieta e/ou exercício, alteraram a resposta autonómica para uma hegemonia do parassimpático, sendo que apenas uma refeição, independente do tipo, influencia também esta resposta. A avaliação destas alterações numa população mais alargada e de forma controlada, permitirá suportar o provável impacto das alterações do sistema nervoso autónomo na disfunção metabólica.

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List of tables

Table 1. Summary of subjects and study design ... 45 Table 2. Primary and secondary outcomes accordingly to the moment of evaluation and study design. ... 49 Table 3. Characterization of meals nutritional composition for Mediterranean Meal (MdM) and Fast Food Meal (FFM) ... 51 Table 4. Summary of the study outcomes and instruments ... 56 Table 5. Allostatic load biomarkers accordingly to the five-physiological system classification and cut-points used accordingly to quartiles or available clinical high-risk scores. ... 59 Table 6. Cyranose ® settings for urine analysis... 64 Table 7. Baseline characteristics accordingly to allocation of intervention order Mediterranean and then Fast Food (MdM-FFM) or Fast food followed by Mediterranean meal (FFM-MdM). ... 73 Table 8. Exercise challenge evaluated outcomes after each of the meals Mediterranean Meal (MDM) and Fast Food Meal (FFM)... 74 Table 9. Baseline demographic and clinical characteristics of the exercise training group (EG) and the control group (CG) that completed protocol. ... 75 Table 10. Baseline, demographic and clinical characteristics of swimmers included (n=27). .... 76 Table 11. Changes blood cell counts before and after exercise with a pre-exercise fast food meal (FFM) or Mediterranean meal (MdM). ... 78 Table 12. Changes in lymphocyte subpopulations relative and absolute number before and after exercise with a pre-exercise fast food meal (FFM) or Mediterranean meal (MdM). ... 79 Table 13. Changes in regulatory T cells numbers before and after exercise with a pre-exercise fast food meal (FFM) or Mediterranean meal (MdM) and in mean fluorescence intensity (MFI) for Foxp3, CD25 and CD127. ... 80 Table 14. Effect of the intervention in physical activity, comparison between usual physical activity (PA) measured during run-in period and during induced sedentary behavior intervention, reduced PA period (n=39) ... 82

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23 Table 15. Effect of the physical activity (PA) reduction in lymphocyte subsets relative percentage in all participants and in those that reduced physical activity by 30%. ... 83 Table 16. Changes in regulatory T cells relative absolute number before and after physical activity reduction in all participants and those that reduced physical activity (PA) over 30% on step number. ... 84 Table 17. Comparison between Mediterranean Meal (MdM) and Fast Food Meal (FFM) effects on ALIq (allostatic load index based on quartiles) and ALIclin (allostatic load index based on clinical parameters) before and after each meal and after exercise challenge (EC) (n=41) ... 85 Table 18. Linear mixed model results evaluating the effect of each meal and evaluation before and after meal and after exercise challenge on allostatic load index. ... 86 Table 19. Allostatic load index changes before and after inactivity. ... 87 Table 20. Multiple linear regression for predicting ΔALIq and ΔALIclin in participants that were able to reduce more than 30% their number of steps (n=27)... 87 Table 21. Comparison between Mediterranean Meal(MdM) and Fast Food Meal (FFM) effects on serum adipokine response before and after each meal and after exercise challenge (EC) (n=39) ... 89 Table 22. Correlation coefficients matrix between adipokines responses to Mediterranean Meal (MdM) and Fast Food Meal (FFM) and exercise challenge. ... 91 Table 23. Odds Ratio (OR) for respiratory infections in the exercise and control groups (data presented as OR [95%CI]). ... 92 Table 24. Metabolites retained in the calibration model were calculated using partial least squares regression with cross-validation for the prediction of the number of respiratory infections in all individuals according to samples collected at the end of the study. ... 94 Table 25. Respiratory infection number and characterization in swimmers that completed follow-up protocol (n=24) ... 95 Table 26. Odds Ratio (OR) for respiratory infections (n=24). ... 95 Table 27. Comparison between fast food (FFM) and Mediterranean (MdM) meals for autonomic nervous system outcomes. ... 98

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List of figures

Figure 1. Study design protocol and participant’s intervention distribution of the randomized crossover clinical trial (Fast food meal (FFM) and Mediterranean meal (MdM)). ... 47 Figure 2. Study design of pilot trial assessing physical activity reduction. ... 48 Figure 3. Flowchart describing the flow of the participants of controlled clinical trial assessing respiratory infections in older adults ... 53 Figure 4. Flow-chart and follow-up of swimmers (URTI-upper respiratory tract infections). ... 77 Figure 5. Points and connecting dots with error bar comparing Mediterranean and Fast Food meals effects on lymphocyte subset response before and after exercise using generalized linear mixed model. Data is expressed in marginal mean estimate and 95% CI. ... 81 Figure 6. Points and connecting dots with error bar comparing Mediterranean and Fast Food meals effects on adipokine response to meal and exercise challenge using linear mixed model. Data is expressed in marginal mean estimate and 95% CI. EC- Exercise challenge. ... 90 Figure 7. Serum targeted metabolomic profiles for both groups, before and after the study period, assessing the effect of exercise training on the exercise and control groups. ANOVA-simultaneous component analysis has shown that differences between all 4 groups are significant with p<0.001. ... 93 Figure 8. Spatial distribution of eNose sensors resistance values based on two components after principal component analysis (PCA). Group 1- cluster 1 and group 2- cluster 2. ... 96 Figure 9. Periodization of the swimming training period and surveillance of respiratory infections episodes. ... 97 Figure 10. Comparing Mediterranean (MdM) and Fast Food (FFM) meals effects on maximal diameter and absolute constriction amplitude. Linear mixed model adjusted for age, gender and body mass index. ... 99 Figure 11. Meta-analysis of muscle sympathetic nerve activity (MSNA) burst frequency (top), MSNA burst incidence (middle) and standard deviation of normal-to-normal intervals (bottom) changes with weight loss. ... 103 Figure 12. Effect of exercise load on the illness risk mediated by immune and metabolic response, coordinated by the autonomic nervous system. ... 121

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1. Introduction

The risk for noncommunicable chronic diseases is influenced by lifestyle factors among which exercise and sedentary behavior are major determinants [1]. Hence, physical activity has been highly recommended and frequently prescribed to control and prevent a range of diseases and conditions such as diabetes or hypertension [2]. However, previous studies have mainly focused on the assessment of exercise, alone or combined with weight management strategies, on health outcomes. The effects of diet and physical activity on health often interact. Indeed, diet modulates immune function and inflammatory processes [3, 4]. Still, effects of an overall dietary approach and of physical activity levels on the immune response to exercise remains poorly known.

Exercise is an important modulator of the immune response. Lymphocyte proliferation is suppressed by acute strenuous exercise [5]. Also, a reduction in numbers of peripheral blood Th1 cell and production of IFN-γ occurs in parallel with an increase in blood Th2 and regulatory T-cells with prolonged and exhaustive exercise [6]. This is clinically translated by an increased risk of illness by very high and no, or low, training bouts [7]. On this context, nutritional strategies have been suggested as potential countermeasures to exercise-induced immunodepression [8]. Dietary supplements, namely carbohydrate ingestion may blunt of the inhibitory effects of exercise on T-cell proliferation and neutrophil phagocytosis/oxidative burst activity [9, 10]. The intake of fruit and vegetables has been correlated to an increase of oxidative capacity [11]. Nevertheless, insufficient studies have address the potential benefits of using whole food versus supplements in immune and health-related outcomes [12].

Mediterranean diet has antioxidant and anti-inflammatory properties [13] suggesting it may be used to modulate immune response to exercise. In contrast, a recent systematic review, demonstrated that a high fat meal was associated with acute inflammatory response, mediated by an IL-6 response [14]; on the other hand a Mediterranean meal reduced blood markers of inflammation [15]. Nevertheless, these meals types where never compared in their interactions with an exercise bout. Response to exercise is not only dependent on diet but also on individual non-modifiable determinants such as genetic, gender, physiologic and psychological circumstances [8, 9, 16, 17]. This reaction can be interpreted within the exposome concept that includes all environmental exposures that a person experiences throughout the life course [18]. The dynamic process of adjusting to homeostasis challenges is referred as allostasis [19]. Allostatic load is a complex clinical construct that takes in consideration the repeated stress and wear-and-tear on the body and brain [19, 20]. This allostatic load concept reinforces the need to interpret exposures within an holistic context and the possibility to modulate these responses with an ultimate goal to improve health.

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26 Low levels of physical inactivity may be associated with low grade systemic inflammation [21] and longer periods of immunodepression [22]. It has been speculated that individuals with a sedentary behavior and those performing high intensity exercise might be more susceptible to illness[3]. Although much effort has been devoted to examining responses to physical activity changes, no holistic metric to measure sedentarism related immune dysfunction has been applied within exercise physiology. Most of the previous literature have focused on metabolic dysregulation biomarkers and negative impact on body composition [23-25], but the acute immune impact has been poorly addressed. Other specific populations namely obese and asthmatic have an intrinsic relation with these changes [26, 27] and their pro-inflammatory state can promote different immune responses. Aging has been associated with an increased susceptibility to infections, mediated by “immunosenescence” and “inflammaging” [28]. In healthy older adults, regular aerobic exercise has been shown to improve adaptive responses, chronic inflammation [29, 30] and mucosal immunity [31]. Lipid peroxidation increases with age and it might be prevented by lifelong aerobic training [32], but also with resistance training [33].

Another specific population commonly studied in their susceptibility to respiratory infections are elite athletes. Respiratory infections in elite athletes might be correlated with intensity of training and has been associated with a specific sport [34, 35], and swimmers seems to present an increased risk [36]. Main reasons are not fully understood, but environment exposure might have a role [37] since chloramine and dysinfection by-products in swimming pools are know to induce airway damage and inflammation and probably modulate the autonomic nervous system response [38].

The autonomic nervous system coordenates the immune response to acute and long-term exercise. Moving from a sedentary to an active lifestyle leads to a reduction of sympathetic nervous system activation [39]. In athletes, higher training loads result in enhanced sympathetic activation [40]. Overweight and obesity, consequences of an unhealthy lifestyle and sedentary habits, have been associated with an increased sympathetic and decreased parasympathetic activity [41-43]. Diet can also influence the autonomic nervous system response, namely Mediterranean dietary pattern has been associated with higher heart rate variability [44]. So, since diet and exercise-based weight loss are the mainstream therapy for obesity; understanding its effect on the autonomic nervous system would help to clarify its potential role as a new treatment target.

Therefore, this study aimed to understand the immune, metabolic, inflammatory and impact of acute exercise and short- and long-term changes in physical activity. Further we aimed to evaluate the effect of a meal, Mediterranean versus a fast food like meals, and diet in the modulation of this stress response to further understand how autonomic nervous system links these immune-mediated responses.

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2.Review of the literature

2.1. Exercise and physical activity

Exercise, physical activity and physical fitness are different concepts, however they are frequently used interchangeably [45]. The Centers for Disease Control and Prevention defines exercise as a type of physical activity that involves a planned, structured and repetitive body movement done to maintain or improve physical fitness [46]. The World Health Organization (WHO) defined physical activity as any bodily movement that results in energy expenditure and also includes exercise as a subcategory of physical activity [47].

As proposed by Gabriel P. et al. [48] physical activity is a complex and multidimensional behavior that balances occupation, transport, leisure and household activities with sedentary behavior producing human movement. In a recent position statement, intensity can be divided in sedentary, light, moderate, vigorous and high[49]. Physical fitness is a group of attributes that people have or achieve. Being physically fit has been defined as “the ability to carry out daily tasks with vigor and alertness, without undue fatigue and with ample energy to enjoy leisure-time pursuits and to meet unforeseen emergencies" [45]. Fitness falls in two main components: health related (including cardiorespiratory, muscular endurance, muscular strength, body composition and flexibility) and related to the athletic ability [45]. It is suggested that in individuals who are unfit/physically inactive, just a small change in physical activity leads to a significant improvement in health status, like a reduction of chronic disease or premature death[50].

Insufficient physical activity is a leading risk factor for non-communicable disease [1] and, furthermore an important impact on mental health and quality of life [51]. One in four adults do not meet the WHO recommendations on physical activity [52]. WHO launched a global action Plan for Physical activity 2018-2030 with the global target of reducing physical inactivity by 10% by 2025 [2]. However, the acute impact of physical inactivity has been rarely a subject of research and although it poses important issues on high level athletes that need to acutely stop their high intensity training due to a lesion or illness (e.g. respiratory infections), but also in daily life. This group of highly trained individuals, that submit themselves to high loads of exercise appear to have a potential attenuation in their health status [50]. In a recent statement by the International Olympic Committee consensus proposes different relations of training loads and risk of illness, suggesting that this risk differs between recreational, sub-elite athletes and elite athletes [7]. However, several unknown factors might contribute to this relationship, particularly the individual immune and metabolic response to exercise.

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2.2. Exercise induced immune changes

Acute or chronic exercise induce different changes in both the cell number and function of the immune system [16], namely in mucosal immunity, innate (e.g. neutrophils, monocytes and natural killer cells) and acquired immune system (T and B lymphocytes) responses [30]. It is agreed that during and immediately after exercise there is an increase in lymphocyte number, proportional to the exercise intensity and duration, falling below pre-exercise levels in the early stage of recovery [16]. However, the clinical significance of these acute changes and the impact on continuous training are still unknown [53]. Furthermore, there is no unique biomarker that truly measures the immune function in a meaningful way. Currently, the main studied clinical outcome of exercise impact on the immunological response is the susceptibility to infections, particularly respiratory infections [3].

The immune and metabolic response to exercise mainly depends on its intensity, and J-shaped model relationship has been proposed [3, 16]. This model suggests that moderate exercise is associated with a lower risk of illness or respiratory infections compared to sedentary or high intensity levels of physical activity, which may raise the respiratory infections/illness risk. Nevertheless, in a recent systematic review, it was not possible to establish if exercise could be interfering with the severity or duration of acute respiratory infections[54]. It has been hypothesized that other factors might be considered in this model, namely physical fitness, concomitant presence of allergy, inflammatory disorders, oxidative stress levels, environmental factors and nutritional status [3, 16, 55]. The sum of these characteristics could then define an illness prone profile and represent a link between the metabolic and immune effects of exercise. Exercise has also an immunomodulating role on metabolic disease, widely recognized by the association of physical inactivity is associated to metabolic disease. Exercise induces relevant anti-inflammatory effects in the context of obesity, as it limits lipid accumulation, inhibits the expansion of adipose tissue and decreases the recruitment of proinflammatory macrophages and CD8+ T lymphocytes, cells known to promote insulin resistance[56].

Recently, a constrained total energy expenditure model [57] was proposed linking metabolic, immune and autonomic parameters. Pontzer et al[58] suggested that humans develop mechanisms to maintain total energy expenditure within a narrow range, dynamically compensating change in physical activity to keep daily expenditure. This occurs at long-term level and explains why weight loss is more significant at higher levels of physical activity. Changing from a sedentary to a more active lifestyle leads to downregulation of non-essential mechanisms, reducing inflammation, autonomic nervous system reactivity that at long term lead to a reduced risk of chronic disease. On the other side, extreme levels of physical activity in which essential function are compromised might negatively affects health. This relationship might be different in the context of a particular disease and/or specific individual characteristics,

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29 namely asthmatic or obese individuals. In fact, both conditions benefit from physical training[27], and their chronic inflammatory profiles have been linked to an increase susceptibility to infections, namely respiratory tract infections[59, 60]. In asthma it has been pointed that this susceptibility could be an epiphenomenon, as infections might appear more severe in patients with underlying respiratory inflammation, or that this increased risk could reflect an altered host susceptibility[60]. Nevertheless, in a mouse model of allergic asthma, aerobic training reduced the local peribronchial activation of leukocytes, airway inflammation (reduced expression of IL-4, IL-5 and IL13) and decreased Th2 response[61]. Except for exercise induced bronchoconstriction evaluation, acute exercise challenge immune and metabolic effects in an individual with asthma have been sparsely reported. Recently, a study showed that airway inflammation response to acute exercise depends on the physical fitness of the individual. Those which were unfit had a decreased eNO after acute exercise challenge than the most fit ones, concluding that the attenuated response in the more physically active could be secondary to a sustained anti-inflammatory effect of exercise training [62]. It is possible that these metabolic and immune response will mainly depend on the duration and intensity of exercise.

2.2.1. Acute bout of exercise

In response to acute exercise an immediate increase in blood leukocyte subpopulations occurs, namely neutrophils and lymphocytes. This increase is correlated with the release of stress hormones, like norepinephrine, epinephrine and cortisol. These effects are mainly dependent on the expression of beta-adrenoreceptor on T, B and NK cells [63]. Furthermore, exercise increases the metabolic rate, oxidative stress and cortisol, that, in turn, modulate the innate and adaptive immune function[16].

In the immediate innate immune response to exercise there is an increase of neutrophils, namely immature neutrophils and monocytes, particularly pro-inflammatory monocytes. Functionally, these responses are correlated with a decrease on neutrophil respiratory burst and degranulation[16]. In a recent systematic review, data on the NK response were inconsistent, although a tendency was seen that favored an increase of NK cytotoxicity after more intense exercise[64]. These profound and transient time-dependent changes also associated with changes on the phenotype and functional capacity of lymphocytes[16].

The initial increase in lymphocyte is mainly regulated by a higher increase of effector and cytotoxic cells (CD8+), leading to a decrease ratio in CD4/CD8. This larger relative difference of CD8+ might be explained by the higher density of beta 2 adrenoreceptors[65]. Lymphocyte mobilization mirrors the differential cellular expression of these receptors, which

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30 are more intense in natural killer cells and CD8+T cells versus B and CD4+ cells. The regulatory T cell response to exercise have showed contradictory results, mainly dependent on the intensity and duration of the exercise bout, with short term bouts with maximal power outputs associating with higher increases of cell number[65]. Exercise also modulates the cytokine populations balance, inducing an impaired IFNγ production, without a consistent effect on IL-10[65]. However, the main cytokine response occurs in IL-6 with concentrations that increase up to 10 fold[65, 66]. Skeletal muscle is one of the major sources of IL-6, a myokine, but also adipose-tissue derived IL-6 can contribute to its systemic increase with exercise[67]. IL-8 and IL-15 are other two important myokines, that, in association with IL-6, can contribute to a Th1 immune response and have a role in metabolism, modulation of insulin sensitivity and glycemic control[68]. Furthermore, cytokines released from muscle cells may trigger adipokines release from the adipose tissue[69]. This supports a potential contribution of metabolic response to the dynamic exercise-associated change in immune cell numbers and function.

The rapid increase of neutrophils and lymphocytes is followed by a return or decrease to pre-exercise levels. This observation led to a proposal that exercise could induce a short-term window of immune suppression, called the “open-window” hypothesis [16]. In a recent systematic review, acute bouts of exercise generated a slight but suppressive effect on lymphocyte proliferation, which were most correlated with longer duration of exercise [70]. Limited quality of evidence has for long supported the claim that vigorous exercise is associated with a clinical meaningful immunodepression [53]. The intense reductions in lymphocyte numbers and function that occur after exercise may in fact reflect a transient and time-dependent redistribution of immune cells to peripheral tissues [71], resulting in a heightened state of immune surveillance and immune regulation, as opposed to immune depression [72]. The mucosal response, measured by secretory IgA (sIgA) is variable and influenced not only by exercise intensity and duration but also by individual fitness [16]. Although sympathetic nervous system modulates IgA synthesis, the acute release of catecholamines may up-regulate expression or mobilization of transepithelial transport of the polymeric immunoglobulin receptor (pIgR)-IgA complex and increase transcytosis of sIgA [9, 16].

When assessing the specific phenotypes of cells preferentially mobilized during exercise, and regarding NK cells, the most exercise-responsive lymphocyte subset are mature NK, which are more capable of rapid effector functions, and previously associated with an increase surveillance to neoplastic cells [73]. Recently, use of acute exercise immune cell mobilization was proposed in cancer treatment [74]. However, several factors might contribute to this response namely the duration of stress, timing, exposure, but also age, gender and genetics that can modulate the response and both immune protector and immune pathological response. Long-term exercise seems also to induce different responses.

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2.2.2. Chronic long-term exercise

Regular exercise exerts both positive and negative effects on the immune system. Most of the studies have focused on increased risk of respiratory infections, due to the observation of an increase number of respiratory infections in elite endurance athletes[9, 16]. Respiratory infections, have been linked to an increase in lipid peroxidation biomarkers[75]. One of the main issues regarding respiratory infections evaluation are the diagnostic confirmation, as symptoms can occur without the laboratorial identification of pathogens [76]. Recently, an hypothesis has been suggested that the increase frequency of respiratory infection in athletes would be incompatible with the training load of high international level athletes, therefore other factors like genetic but also behavioral might explain why international level athletes suffer from less respiratory infections than national level ones [30, 36]. Outside the high-level athlete, exercise due to its general health benefits may be associated with a decrease of severity and duration of acute respiratory infections. In a systematic review of the literature, evidence was not consistent to determine exercise as effective in altering the occurrence, severity and duration of acute respiratory infections[54].

Despite that blood leukocyte counts may change with an exercise bout, moderate intensity exercise seems to induce a beneficial effect on immune function[9, 16]. Long term moderate exercise induces innate immune changes with a decrease in monocyte´s inflammatory phenotype[16], an increase neutrophil phagocytic activity[77] and also greater NK cell cytotoxic activity [9]. Body fat mass reduction after a combined intervention of exercise and diet led to a reactivation of NK functionality, showed by an increase of interferon gamma expression in CD 56dim_{NK cells [78]. With regular exercise there is also a reduction in} expression of pro-inflammatory TLRs in monocytes and macrophages[79].

Regular moderate intensity exercise has also been linked with exercise-induced immune enhancement, particularly in the acquired immune responses, namely with an increase vaccinal responses [80, 81], a reduced number of exhausted and senescent T cells, increase on T-cell proliferation and decrease of circulating inflammatory cytokines [9]. These benefits, partially mediated by a circulating IL-6 and increase expression of IL-10, have been correlated with positive effects on body composition [82]. Reduction in visceral fat mass can have also an indirect beneficial effect, through the reduction of pro-inflammatory adipokine secretion and a switching from M1 type inflammatory macrophages to M2 type anti-inflammatory macrophages, that induce a higher expression of IL-10 and adiponectin [79]. The moderate acute elevation of both IL-6 and IL-10 induced by moderate exercise may cause an inhibition of TNF alfa and stimulate IL-1 ra, limiting IL-1 β, a pro-inflammatory mediator of beta cell damage in type 2 diabetes [83]. Further, IL-6 has a direct effect in insulin sensitivity and fat oxidation[83]. Finally exercise increases the antioxidant defense system. In this way, exercise seems to be a strong anti-inflammatory and metabolism-improving strategy [83], but response can vary in specific

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32 populations. Elderly are a specific population whose exercise has been promoted to improve immune and health outcomes.

Nevertheless, excessive, high intensity, high-volume exercise can, in opposition, contribute to depression of the immune system and increase the infection risk. Several factors can interfere with this relation, namely physical and psychological stress, and pathogen exposure[9]. Repeated exercise bouts and extended periods of intense training are specific of the high level athlete population and can be associated with overreaching where the immune system does not recover from stress related exercise impact; usually this is also associated with environmental factors (thermal stress, malnutrition) leading to an impact on immune function and increase susceptibility to opportunistic infection[84]. This correlation of different factors will be further addressed in the high-level athlete model.

2.2.2.1 Exercise and older adults

Aging is associated with an increased susceptibility to infections, cancer, cardiovascular and neurodegenerative diseases. Age-associated immune dysfunction, “immunosenescence” and

“inflammaging” have an impact on the development of these chronic diseases and can also increase the risk of infection [28]. Immunosenescence is characterized by imbalance of naïve and memory T cell subpopulations caused by thymic involution, by low grade inflammation (associated with an increase expression of IL-6, TNF-a and C-reactive protein), accumulation of senescent T cells and telomere shortening and resistance to apoptosis [85]. Oxidative stress plays a major role in low grade inflammation, a characteristic of age-associated diseases [86]. Oxidative stress theory of aging stipulates that reactive oxygen species, like peroxides and aldehydes, play a role in oxidative damage to cells and in the imbalance of pro-oxidants and antioxidants, resulting in apoptosis or a progressive decrease in cellular function [87, 88], which lead to immunosenescence [86]. However, evidence of this relationship is still controversial [87]. Lipid peroxidation increases significantly with age, and this increase seems to be prevented by lifelong aerobic exercise training[32], but also with resistance training[33].

Sedentary behavior in older adults significantly affects physical performance[89]. In healthy older adults, regular aerobic exercise has been shown to improve adaptive responses, chronic inflammation [29, 30] and mucosal immunity[31]. In a recent systematic review exercise modulated markers of cellular immunosenescence, namely an increase of naïve T cells in peripheral blood at rest[90]. In a clinical trial evaluating moderate-intensity exercise training in postmenopausal women, the actual incidence of common colds was reduced [91]; this was reproduced in a large community study where the number of respiratory infections and infection-related burden decreased [92].

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33 Recently, an anti-immunosenescence effect of exercise has been explained by the proposed “exercise immunological space hypothesis” that is based on three parameters: (1) late-stage differentiated phenotype cells that are mobilized to peripherical blood during exercise; (2) apoptosis of these cells in the inflamed tissues and (3) naïve T cell repertoire expanding in response to this created immunological space[93]. Another counter-acting measure to T cell immunosenescence can also occur indirectly, as an active lifestyle limits adipose tissue accumulation, reducing the aging and obesity related dysfunction. However, conflicting results have been reported regarding the association of aging, exercise and oxidative stress [94], as in elderly populations, immunological and oxidative stress responses to exercise can be different [95]. To this difference also contributes the life-time “dose” of exercise, when assessing individuals that are exposed to moderate doses of exercise. It is likely that exercise promotes anti-immunosenescence effect in a dose-dependent manner and until a threshold, as training influences immunological response[96]. At extreme conditions and high level exercise, immunosenescent profiles might be exacerbated, due to a dysregulation of redox hemostasis, an excessive inflammatory response that leads to inflammation and can correlate with viral reactivation[93].

2.2.2.2 The high-level athlete

Athletes have been described as a respiratory infections susceptible population [30], which may have a relevant impact in their performance in high competition challenges. In a four years prospective cohort of 28 professional swimmers, a significant increase in respiratory infection risk for every 10% increase in resistance and high-load trainings was observed[36].

The “open window theory” of altered immunity has been used to explain the athletes susceptibility[97]. This open window hypothesis is believed to occur after repeated bouts of acute strenuous exercise that are performed without adequate recovery. It was demonstrated that after each bout of prolonged exercise a period of 3 to 72 hours of altered immunity can occur[98]. If a second bout is performed during this window, the exercise-induced enhancement in immunity is blunted and the post exercise immune depression is more severe and prolonged[9]. Several main issues can be raised in order to understand if an athlete is an immune-compromised host, if there is a role of intense and prolonged exercise training in their increased susceptibility or if other risk factors might be involved, namely stress, environment or nutrition[3, 4, 99]. Peake et al., reviewed the effect of repeated exercise bouts and extended periods of training, arguing that, despite contradictory results, several studies associated with functional overreaching found a more significant decrease in neutrophil degranulation, lymphocyte proliferation and antibody production [84].

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34 Recently, Gleeson et al. studied potential factors that could predict the increased risk for upper respiratory infections in athletes, evaluating a multicomponent immune model including clinical and immunogenetic risk, the status of Epstein Barr virus infection, salivary IgA and the inflammatory response to short and long term exercise; he showed that low levels of sIgA in pre-exercise, viral detection of EBV DNA in saliva and a genetic predisposition for pro-inflammatory cytokine response were associated with higher risk for respiratory infections[100]. Another specific observation is the difference between national and international levels swimmers in the risk of respiratory infections, where in a 4 year follow-up cohort the risk for respiratory infections where higher at national than international level swimmers [36]. Therefore, specific behavior factors, particularly the association of physical and psychological challenges could influence the neuro-endocrine-immune modulation response to exercise [101]. In a recent study, level of anxiety status prior to exercise modulated in vivo immune response after exercise [102].Several strategies have also been studied and proposed to modulate immune response to stress in athletes, particularly using nutritional strategies and supplements [101, 103].

2.3. Impact of physical inactivity

Acute exercise effects have seldom been studied in populations other than athletes or healthy physical active populations [97, 104, 105]. Mills et al. [17] proposed that being physically fit is protective of inflammatory responses. High physical activity levels have been associated with lower inflammation [106] and reduced risk of cardiovascular disease [107]. In a recent study moderate-to-vigorous physical activity has been associated with a favorable profile of inflammatory biomarkers, independent of relevant cardiometabolic disease risk factors [108]. Most chronic diseases, particularly metabolic and cardiovascular disorders, are influenced by physical inactivity. Recently, patients with symptomatic chronic heart failure who engaged in more sedentary behavior were found to have a higher mortality rate[109].Possibly, reducing the physical activity, using a reverse logic, might also be associated with a decreased immune function response.

New studies have emerged studying the free-living physical inactivity [110], using real-life scenarios, through a reduced ambulatory activity by number of daily step reductions. Previous evidence of bedrest has been associated with an impairment of lipid trafficking and insulin resistance [111]. Biochemical and molecular mechanisms of physical inactivity are not simply the converse of physical activity [51]. Pedersen and coworkers showed that a short reduction of physical activity reduced peripheral insulin sensitivity, caused changes in body composition with an increase in total fat percentage and total body mass, and reduced cardiovascular fitness evaluated by VO2max [23-25, 98]. Similar results were seen by Davies et

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35 participants with or without familiar diabetes risk showed similar results [112]. Recently, in older adults it was found that insulin resistance increased after induced sedentary behavior and with the restart of physical activity insulin sensitivity returned to usual levels or better values than before intervention [110, 113]. In older adults a significant impact was also seen with loss of leg muscle mass [114]. The effect of a physical inactivity intervention on immune function have not been studied [23-25].

2.4 Dietary modulation of exercise response

Several nutritional strategies have been used to counteract exercise-induced immunodepression and systemic inflammation, particularly in athletes [8, 26, 84, 115]. Globally and accordingly to a recent consensus statement as long as diet meets the energy demands and provides sufficient macro and micro nutrients to support immune system, there is no need for specific supplements[115].

Nevertheless, diet can influence the immune system, introducing the concept of immunonutrition, which is the potential to modulate the activity of immune system with specific nutrients. This has been studied in several areas, particularly in critical illness where several macronutrients and micronutrients have been studied for its impact/beneficial in protecting immunological consequences of critical illness [116].

Carbohydrate supplementation has for long been recognized to have a role as a

countermeasure against exercise-induced immune impairment, particularly when is consumed during exercise. Glucose acts as a substrate for immune cells and carbohydrate consumption during exercise maintains plasma glucose levels stable, blunting stress hormones response[115], thus reducing the redistribution of neutrophils, monocytes and lymphocytes[84]. An adequate protein intake is also necessary to maintain normal immune function[8], ingesting protein after exercise minimized the lymphocyte redistribution and reduced the risk of respiratory infections[117]. Amino acids have also been studied, particularly glutamine, which is a precursor in the synthesis of nucleic acids and an important fuel for immune cells, however laboratory-based exercise studies did not show consistent beneficial results[115]. Similar results were seen for branched chain amino acids (leucine, isoleucine and valine), as evidence regarding these supplements are not enough to recommend their use[115].

Dietary fats seems to play a role in modulating immune functions and inflammatory

processes[26]. No main differences were seen between high and low fat diet on lymphocyte and leukocyte subsets, neither on cytokine response[118]. Recent studies have further considered the modulation of exercise in the metabolic response to a high-fat diet[119]. A high fat meal

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36 challenge has been associated to an increase of airway inflammation, mediated by neutrophils, and a decrease in airway reversibility in a population of obese individuals with asthma[120]. Specific supplements, namely omega-3 polyunsaturated fatty acids (n-3), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) particularly found in oily fish and fish oils, have been further studied. These supplements showed to have anti-inflammatory effects[115]. However, in the context of exercise, evidence is limited[115].

Exercise leads to an imbalance between the free radicals and reactive oxygen species (ROS) produced and the antioxidant defense system[103]. Immune system activity is one of the main determinants for exercise-generated radical species[115]. However, body normally responds with their endogenous antioxidant defenses. There is some evidence that supports that the intake of relatively high doses of antioxidant vitamins can reduce cortisol and anti-inflammatory cytokine response to prolonged exercise[121]. The use of vitamin C has been associated to a reduced incidence of colds in population exposed to brief periods of severe physical exercise[122], and also linked to a reduction of IL-6 and cortisol response to exercise[8, 103].

Vitamin D has a key role in innate and acquired immunity as well as in the regulation of

immune response[115]. The optimal serum vitamin D concentration, particularly in athletes, is still associated with important controversy. Low vitamin D status is associated with low secretion of IgA in saliva, and increase respiratory risk of infection[8]. However, in a recent study it was showed that athletes with levels over > 50 nmol/l still had a higher risk of respiratory infections comparing to those with levels over 75 nmol/l[123]. Vitamin D supplementation should be planned in athletes who show vitamin deficiency and through regulated protocol decisions, particularly due to the risk of over supplementation[124]. As for another vitamin, namely Vitamin E, or specific minerals as Zinc, there is still no evidence that supplementation will be beneficial[115].

Plant polyphenols, include a class of flavonoids, are associated with strong anti-inflammatory and immune-regulatory properties[115]. They have been studied as countermeasure to exercise-induced physiological stress using purified polyphenol supplements (ex. Quercetin), but also plant extracts (ex. green tree) and increase fruits consumption. Intake of supplements or foods rich with naturally occurring polyphenolic compounds was associated with a lower incidence of upper respiratory infections[125] and short-term anti-oxidant effects[8, 126].

Probiotic-rich foods and supplements contain non-pathogenic bacteria that colonize the gut associated with health benefits[127], as well as modulate immune function[8]. In a systematic review, it was suggested a small, but beneficial effect in trained individuals and a

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37 potential reduced risk of respiratory and gastrointestinal illness during stressful periods of training and competition[128, 129].

2.4.1. From nutritional supplements to a meal

Although several nutrition supplements have been addressed, a balanced and well-diversified diet that meets the energy demands is a key component. However, meals with the same energy content can be very different in micronutrients. Nutrients interaction within a meal might lead to different outcomes. Most of the studies regarding fast food meal and exercise have assessed the potential benefit of exercise in reducing the metabolic and inflammatory negative impact of a fast food [130] or high fat meal[131]. This is correlated with the immediate effects of the meal, particularly those containing high level of fat, that induce an increase of inflammatory markers[132] and cause postprandial lipemia[133]. However, even with meals with similar fat content, as they differed in the type of fat, particularly in different dairy products, differences occurred in postprandial triglyceride responses.[133, 134]. In a recent systematic review, modulating the meal content, namely increasing the polyunsaturated fat in the meals, was associated with improved glycemia, insulin resistance and secretion[135]. Mediterranean meal is particularly rich in polyunsaturated fatty acids[136].

The traditional Mediterranean diet is the dietary pattern prevailing among populations of the olive tree-growing areas of the Mediterranean basin before the mid-1960s, that is, before globalization made its influence on lifestyle[137, 138]. The Mediterranean diet has been linked to a number of health benefits, including reduced mortality risk and lower incidence of cardiovascular disease[139]. Nonetheless, studies have also been confounded, in many instances, by different measures of adherence, durations of intervention and heterogeneity of both participants and outcomes[140]. Although the benefits of adherence to Mediterranean diet have been shown, even for short periods such as four weeks, the metabolic effects of a single meal are not well known [141]. A recent study comparing fast food with organic beef meal or turkey meal showed that a meal with less saturated and trans fatty acids quantity induced a more significant decrease in LDL-cholesterol [142]. Immunologically the use of olive oil and walnut breakfasts were associated with a lower expression of postprandial pro-inflammatory genes[143].

In association with exercise most of the studies evaluating a pre-exercise meal assessed the potential benefit on performance and not on immune and metabolic response[144]. In a recent study evaluating circulating cytokines response to exercise

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38 associated with a pre-exercise meal no significant changes were showed [145]. In a small study in eight healthy runners, a pre-exercise high-carbohydrate meal induced less change in circulating numbers of leucocytes, neutrophils and T lymphocyte subset immediately and 2 hours after exercise in comparison with a low-carbohydrate meal[146]. The use of a pre-exercise specific meal might modulate the immunometabolic response to pre-exercise, particularly if comparing a Mediterranean type of meal, which is linked to anti-inflammatory properties to a fast food meal.

2.5. Allostatic load and stress biomarkers

“For natural selection acts by either now adapting the varying parts of each being to its organic and inorganic conditions of life; or by having adapted them during long past periods of time: the adaptations being aided in some cases by use and disuse, being slightly affected by the direct action of the external conditions of life.”

Charles Darwin in “On the Origin of Species”

As stated by Charles Darwin only organisms that can adapt to a changing environment are able to survive. Walter Canon approached this concept using the term bodily homeostasis, defining it as the result of liberating functions of the nervous systems that adapt the organism to new situations [147, 148]. Hans Selye, founder of modern stress research, described the physiological response to stress as mutual actions of forces that take place across any section of the body, physical or psychological[149].

Stress response includes activation of the autonomic nervous system and of the hypothalamus-pituitary-adrenal axis leading to an increase of catecholamines and corticosteroids[147]. Immune and metabolic systems are also involved as they communicate directly, indirectly and in a non-linear manner contributing to stress. Inefficient response or excessive amount of stress can induce wear and tear of the body and ultimately lead to disease[147].

Allostasis is a closely linked concept that was defined by Sterling and Eyer as “achieving stability through change”[147]. In face of a challenge, the body responds by turning on an allostatic response. If allostatic response is limited to the period of challenge, protection via adaptation predominates over adverse consequences. However, continuous exposure to elevated stress hormones can result in allostatic load and overload[147, 150]. Allostatic state is a response pattern in which physiological systems are overactive and/or dysregulated[151]. If imbalance is prolonged it might affect adequate energy balance and induce allostatic overload[147].

Allostatic load (AL) is used to define the burden that results from individual adaptation to the physiological dysregulation caused by chronic stress [152, 153] and is the cumulative result