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Keywords:

  • Physical activity;
  • appearance evaluation;
  • psychological distress;
  • conditional indirect effect

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Background

Based on previous research, the aim of this cross-sectional study was to examine the indirect effect of physical activity on psychological distress through appearance evaluation. The indirect effect was hypothesized to be conditional on gender, with the effect being more profound among females than among males.

Method

A total of 2055 adolescents (Mage = 15.3) completed a self-report questionnaire.

Results

Physical activity was indirectly related to psychological distress through appearance evaluation in both males and females, but the indirect effect was stronger for females than for males.

Conclusions

Physical activity may prevent distress through enhanced appearance evaluation.

Key Practitioner Message
  • While the evidence of positive mental health outcomes of physical activity among young people in terms of alleviating psychological distress is amounting, the mechanisms underlying these relationships are not well understood
  • One possible explanation of the inverse relationship between physical activity and psychological distress may be related to an increase in peoples' appearance evaluation
  • Findings suggest that participation in physical activity during adolescent years better enables young people to maintain a satisfying level of appearance evaluation, subsequently better preparing them to cope with psychological distressing emotions
  • A better understanding of mechanisms linking physical activity to mental health is important for the development of optimal intervention programs for young people

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Psychological distress in the format of anxiety and depression is a condition that especially affects young people compared with other population groups (Clark, Smith, Neighbors, Skerlec & Randall, 1994; Compas, Orosan & Grant, 1993; Veit & Ware, 1983). It is a widespread belief that physical activity can alleviate psychological distress in young people, and a number of studies have shown an inverse relationship between level of physical activity and depression and anxiety (e.g., Abu-Omar, Rütten & Lehtinen, 2004; Motl, Birnbaum, Kubik & Dishman, 2004).

While the evidence of positive mental health outcomes of physical activity among young people in terms of alleviating mental health risk symptoms and in generating psychological well-being is a mounting (Biddle & Asare, 2011), the mechanisms underlying these relationships are not well understood. Potential third variables may elucidate the relationship between participation and outcomes, and subsequently contribute to further understanding of why or how they are related. Indeed, Johnson and Taliaferro (2011) encourage future studies to explore potential mediating mechanisms that may explain the inverse relationship between physical activity and psychological distress in adolescents.

One possible explanation of the inverse relationship between physical activity and psychological distress may be related to an increase in peoples' self-esteem. For example, Joiner and Tickle (1998) showed that high amount of exercise was related to less depressive symptoms in adult females, partially mediated by general self-esteem. Nevertheless, self-esteem is considered multidimensional, consisting of domain-specific components, such as academic self-esteem, social self-esteem, and physical self-esteem (Harter, 1999; Sonstroem, 1998). Hence, sub-domains should be regarded as being even more easily influenced by external events or behavioral influences (Harter, 1999; Shapka & Keating, 2005). Moreover, while self-esteem is a central component of psychosocial development during adolescence (Harter, 1990; Rosenberg, Schooler, Schoenbach & Rosenberg, 1995), the perceived importance of domain-specific self-perceptions in this age period may well act to alleviate psychological distress if self-perception in that domain is high.

Research has shown physical self-esteem to be important for young peoples' general self-esteem and psychological well-being (Fox, 2000), and perceptions of one's physical self may be particularly sensitive to variations in physical activity level (Bowker, 2006). One of the most frequently reported dimensions of physical self-esteem is perceived physical appearance (Mendelson, Mendelson & White, 2001; Harter, 1990). Intervention studies have shown that exercise and training can reduce appearance dissatisfaction and enhance physical self-perception and body image (Burgess, Grogan & Burwitz, 2006; Campbell & Hausenblas, 2009; Duncan, Al-Nakeeb & Nevill, 2009). Indeed, an increase in motor skills emanating from physical activity involvement may benefit adolescents' psychological well-being through a sense of mastery (Fox, 1999). Nevertheless, enhanced physical appearance may also benefit well-being by alleviating psychological distress. One possible mechanism for the latter may be that being physically active generally leads to positive changes on objective physical parameters, such as weight loss, being leaner, and gaining a more muscular body.

Female adolescents are consistently less satisfied with their physical appearance than their male counterparts (Smolak, 2004).Thus, not surprisingly, most studies on physical appearance have primarily focused on females. Considering the cultural emphasis placed on the female appearance and looks, one may argue that the consequences of negative perception of appearance on perceived psychological distress may be particularly strong in female adolescents. Siegel (2002) pointed to the cultural emphasis on a slim female body figure, paired with the accumulation of body fat during puberty as a central issue regarding psychological distress throughout the maturation years. Furthermore, empirical studies have shown adolescent girls to experience more symptoms of psychological distress than adolescent boys (Ohannessian, Lerner, Lerner & Eye, 1999; Siegel, Yancey, Aneshensel & Schuler, 1999). The attractiveness ideals of the western culture may elicit negative feelings in females because of the perceived importance of appearance. Indeed, especially among females, low appearance schema scores have been found to be related to higher self-report of depression and other mental health risk indices during adolescence (Harter, 1990, 2000; Mendelson et al., 2001; Sinton & Birch, 2006).

While boys may be evaluated based on their motor competence in sport and physical activity and thus more easily benefit from their participation in terms of distress by means of motor proficiency, girls taking part may more constantly be “on-line” in terms of other peoples' viewpoints and gazes on their appearance, physical attractiveness, and outlook. Hence, despite potential gains in terms of physical-motor parameters, they may still be more vulnerable to feelings of distress.

On the other hand, a growing interest in the body-image concerns of males in western cultures in the last decades can be observed. An increasing number of studies report that boys and men are also dissatisfied with their bodies (Adams, Turner & Bucks, 2005; Pope et al., 2000). Negative perceptions of one's physical self may arise from sociocultural pressures for males to be lean and muscular, in addition to the pressure for girls to be thin and slender (Thompson, Heinberg, Altabe & Tantleff-Dunn, 1999). Hence, aspects of body image may be regarded important precursors for positive mental health in both males and females. Indeed, Hausenblas and Symons Downs (2001) found no gender difference in effect size when investigating body-image dimensions in athletes compared with non-athletes. Summing up, while enhanced body satisfaction through sports and physical activity may particularly alleviate psychological distress among girls, boys as well taking part in sports and physical activity may psychologically benefit from feeling good about their appearance (Siegel, 2002).

In this study, we examined the indirect effect of physical activity on psychological distress through physical appearance in male and female adolescents. The indirect effect was hypothesized to be conditional of gender, that is, the association between appearance evaluation and psychological distress being stronger in female adolescents than in males.

From a statistical point of view, one may argue that including relevant covariates will increase the precision in estimation of model coefficients, because all effects are adjusted for the shared association between the covariate(s) and the variables in the model.

Taking into account individual psychological characteristics would seem important when examining mental health gains from physical activity participating. Of relevance in the present case is that not all individuals are expected to be equally concerned about their physical appearance. The strength of one's appearance-specific self-focus may be of importance when considering the mediation role of appearance evaluation in the physical activity – psychological distress link. Appearance orientation is believed to affect body dissatisfaction by heightening focus on, recall of, and incorporation of appearance-relevant feedback (Hargreaves & Tiggemann, 2003). Hence, it would seem important to control for variations in young peoples' appearance orientation when investigating the link between physical activity, appearance evaluation, and psychological distress.

The current study

Based on the research that indicates an inverse association between physical activity and psychological distress in the format of anxiety and depression in adolescents, the aim of this cross-sectional study was to examine whether involvement in physical activity is indirectly and inversely associated with psychological distress through higher level of appearance evaluation, when controlling for appearance orientation and age. In light of previous research, we further expected that the strength of the indirect effect through appearance evaluation would be conditional on gender, with the effect being more profound among females than among males. The hypothesized model is illustrated in Figure 1.

image

Figure 1. The hypothesized moderated mediation model (model 3; Preacher et al., 2007)

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Method

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Participants and procedures

This cross-sectional study was conducted as a part of the “Goodness of fit in Norwegian Youth Sport”-study. The data were collected between October and December 2008. Based on a representative sample drawn by Statistics Norway, stratified according to school level, school size, and geographical area, a total of 2,971 pupils from 38 different secondary and upper-secondary schools in Norway were invited. A total of 2,055 (71%) adolescents (995 boys and 1,060 girls) from the age 13–18 (mean age 15.3 years) completed the questionnaire. The sample included 88% native Norwegians. The pen and paper-based self-report questionnaire was administrated and supervised by the class teacher, and was completed during school time and put in sealed envelopes when finished. Actual sample sizes vary due to missing data. The Norwegian Social Science Data Service (NSD) approved the study, and parents and children signed a written informed consent before participating in the study.

Measures

Psychological distress

The Norwegian translated version of the ten-item Symptom Check List (SCL-10; Strand, Dalgard, Tambs & Rognerud, 2003) was used to measure psychological distress. This abbreviated version of the SCL-25 (Derogatis, Lipman, Rickels, Uhlenhuth & Covi, 1974) consists of four items related to the dimension anxiety and six items for depressed mood. Participants were asked to respond to the items according to their experience during the previous week. Typical items are “Feeling scared for no reason” (anxiety) and “Feeling of worthlessness” (depression). The ten items were answered on a four-point scale ranging from 1 (not at all) to 4 (extremely). The mean score of all items is used as the measure of psychological distress. The SCL-10 has previously been proved sufficiently reliable in Norwegian samples, and is advisable for practical reasons (Strand et al., 2003). Previous validations support the usage of this scale as a general measure of psychological distress (Rosen et al., 2000). Cronbach's alpha in this study was .90, which was considered acceptable (Cortina, 1993).

Body-image dimensions

The Appearance Evaluation (AE) and Appearance Orientation (AO) scales from the Norwegian translation (Loland, 2000) of MBSRQ-AS (Cash, 1994) were assessed. The AE scale consists of seven items measuring individuals' overall subjective appraisal of their appearance and attractiveness. The AO consists of 12 items, and measures cognitive and behavioral investment in one's appearance, degree of importance in being attractive, attending to one's look. Items on both scales are answered according to five-point Likert scales (1 = definitely disagree; 5 = definitely agree). Scale scores are derived by reverse-scoring contraindicative items, summing all items and then calculating mean item scores. High AE scores indicate greater body satisfaction, and high scores on AO indicate greater importance attached to one's appearance. Both measures have received extensive psychometric evaluation (Brown, Cash & Mikulka, 1990; Cash, 1994; Loland, 2000). The cronbach's alpha coefficients for AE and AO were in present study considered acceptable (.79 and .80, respectively) based on the recommendations of Cortina (1993).

Physical activity

The instrument used to measure physical activity level (PA) was taken from Sagatun, Søgaard, Bjertness, Selmer and Heyerdahl (2007). In this study, however, the same question was asked twice and specified to two different physical activity contexts. The first one consisted of club-led organized sport participation and the second included unorganized physical activity outside of club/school in the format of self-initiated physical activity including games play, and exercise activities taking place alone or with others. With regard to the two contexts (sport participation and unorganized physical activity, respectively), participants were asked the question: “How many hours per week do you play or exercise enough to make you sweat or breathe hard?” (categories: 0; 1–2; 3–4; 5–7; 8–10; 11 hrs or more per week). The six original categories were coded 0, 1–5, and the sum score of the two ordinal variables indicated the total amount of physical activity, ranging from 0 to 10. This score was regarded as continuous data. Sagatun (2010) reported that the question correlated fairly well with other measures, and was the one that best predicted physical activity measured using accelerometer.

Statistical analyses

All statistics were calculated using IBM SPSS version 19.0. Means and standard deviations were used when presenting central tendencies and dispersion. Independent t-tests were used when testing potential gender differences in major study variables (Table 1). When examining potential correlations between major study variables (Table 1), Pearson's product-moment correlation coefficient was used.

Table 1. Descriptive statistics for and correlations among study variables according to gender
 MalesFemales12345
  1. Notes: Boys' correlation coefficients are below the diagonal, girls' correlation coefficients are above the diagonal. PA, Physical Activity level (scale 0–10); AO, Appearance Orientation (scale 1–5); AE, Appearance Evaluation (scale 1–5); Distress, Psychological distress in the format of anxiety and depression (scale 1–4);

  2. a

    Statistically significant higher values compared with opposite gender; independent sample t-test (p < .01).

  3. b

    statistically significant, p < .05;

  4. c

    statistically significant, p < .01.

n9951,060     
1 Age15.3 (1.51)15.3 (1.51)−.16c−.09c−.11c.20c
2 PA3.9 (2.66)a3.3 (2.28)−.15c.04.14c−.06b
3 AO3.2 (0.69)3.5 (.65)a−.02.10c−.03.02
4 AE3.5 (0.82)a3.1 (0.84)−.01.16c.21c−.37c
5 Distress1.5 (0.57)1.8 (.69)a.10c−.10b.06−.17c

Because this study consisted of individuals from 38 different schools in Norway, the possibility of clustering effect on school-level existed. The variables investigated in the model were hypothesized to be student-level constructs, and therefore the possibility of school-level variance was assumed to be low (Martin, Bobis, Anderson, Way & Vellar, 2011). However, to take into account the possible non-independence produced by school variation in levels of the variables, analyses were performed with school included as 37 dummy variables and treated as covariates.

To test for indirect effects when also controlling for covariates, we used the Preacher and Hayes (2008) bootstrapping technique for mediation analysis. Bootstrapping is a nonparametric resampling procedure that can be used to test the null hypothesis for an indirect effect. In comparison with other procedures, this method does not rely on the assumption that the total and indirect effects are normally distributed (Preacher & Hayes, 2008). This procedure (Preacher & Hayes, 2008) allows us to examine a simple mediator model with the inclusion of covariates. In addition to normal theory regression procedures, this technique produces point estimate and bias-corrected (BC) confidence interval for the indirect effect. A BC confidence interval that does not include zero indicates a statistically significant mediation. The number of bootstrap samples is 20,000.

When the strength of the relationship between two variables is conditional of a third variable, moderation is said to be occurring (Preacher, Rucker & Hayes, 2007). When testing the indirect effect conditional of gender, we used the MODMED-macro (model 3) for SPSS (Preacher et al., 2007). This procedure investigates the indirect effect of physical activity on psychological distress through appearance evaluation conditional of gender, that is, the effect of appearance evaluation is moderated by gender, and produces point estimates and BC confidence intervals for specific values of the moderator (in this case, males coded 1 and females coded 2).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Gender differences were found for all observed variables except for age. Girls reported higher prevalence of psychological distress, higher level of investment in one's appearance, and lower satisfaction with their own appearance when compared with boys (Table 1).

Bivariate correlations of the measured variables are reported by gender (Table 1). In general, the observed relationships between the variables were low-to-moderate. The strongest association was the correlation between appearance evaluation and psychological distress in females (r = −.37, p < .01).

The model explained 16% of the variance in psychological distress. As can be seen in Table 2, physical activity positively predicted appearance evaluation (a path). Furthermore, appearance evaluation negatively predicted psychological distress (b path). The model also revealed a significant total effect (c path) of physical activity on psychological distress. Appearance evaluation was inversely related to psychological distress in the model (b path). When investigating the indirect effect of physical activity on psychological distress through appearance evaluation (ab path), the BC confidence interval did not include zero. This suggested an indirect effect of physical activity on psychological distress through appearance evaluation. In other words, higher level of physical activity predicted more positive appearance evaluation, which in turn predicted lower levels of psychological distress. The covariate appearance orientation also individually predicted level of distress. In other words, individuals who reported higher importance put on appearance also typically reported higher level of perceived distress.

Table 2. Mediation analysis for physical activity on psychological distress through appearance evaluation
 CoeffSENT pBC 95% CI
LowerUpper
  1. Notes: Dependent variable: Psychological distress. Coeff, Point estimate of effects; SE, Standard error of the point estimate; NT p, Normal theory p-value; BC 95% CI, Bias corrected 95% confidence interval; PA (IV), Physical activity level; AE (M), Appearance evaluation; CV, Covariates; AO, Appearance Orientation.

  2. Coefficients of School-covariates not shown, for clarity purposes.

PA (IV)
IV to M (a path).057.009<.01  
Total effect (c path)−.016.007.01  
Direct effect (c′path)−.003.006.60  
AE (M)
Direct effect (b path)−.224.018<.01  
Indirect effect (ab path)−.014.003 −.018−.009
Partial effect of CV
Age.050.038.19  
AO.079.022<.01  
Model summary (R2).16 (<.01)

When investigating the moderating effect of gender in the relationship between appearance evaluation and psychological distress (Table 3), the significant interaction indicated a moderated mediation. In other words, the indirect effect of physical activity on psychological distress through appearance evaluation is conditional of gender. Further investigation of the conditional indirect effect revealed significant point estimates in both males and females, indicating that the indirect effect is evident in both genders. However, the indirect effect is stronger in females than in males.

Table 3. Testing for conditional indirect effect
  Mediator modelOutcome model
PE (SE)PE (SE)BC 95% CI
  1. Notes: Dependent variable: Psychological distress. PA, Physical activity level; AE, Appearance evaluation. PE (SE), point estimate and standard error. Conditional indirect effects are bootstrap-generated point estimates, and bias corrected 95% confidence interval (BC 95% CI) when moderated mediation is detected.

  2. a

    denotes statistically significant PE (p < .01). Coefficients of covariates not shown, for clarity purposes. Covariates are AO, age, and School.

PredictorPA.056 (.009)**.001 (.006) 
MediatorAE −.045 (.059) 
ModeratorGender −.518 (.127)a 
Interaction −.103 (.036)a 
Conditional indirect effectMale −.008 (.002)a[−.013, −.005]
Female −.014 (.003)a[−.020, −.009]

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

To the best of our knowledge, no study has previously investigated the conditional indirect effect of physical activity on psychological distress through appearance evaluation moderated by gender in a sample of adolescents. Consistent with previous research (Dishman et al., 2006) the findings support the mediation part of the hypothesized model that higher level of physical activity is inversely and indirectly related to psychological distress through enhanced appearance evaluation in adolescents. Physical activity is probably followed by enhanced physical fitness including aerobic capacity and muscle strength, weight loss, and feelings of a more “toned” body (Biddle & Mutrie, 2007), which in turn may positively affect the way individuals perceive their own appearance. Feeling good about one's “outer self” in the format of a positive evaluation of one's physical appearance may in turn help alleviate the level of psychological distress by fueling one's general self-esteem (Fox, 2000; Harter, 1990; Sonstroem, 1997). The mediation findings revealed even hold when variations in the adolescents' appearance-specific self-focus was controlled for. Findings suggest that participation in physical activity during adolescent years better enables young people to maintain a satisfying level of appearance evaluation, subsequently better preparing them to cope with psychological distressing emotions. In contrast, physically inactive young individuals being in less favor of their “outer self” may more easily come to experience negative affection and feel distressed.

Furthermore, the testing of conditional indirect effect revealed that the mediating effect of physical activity on psychological distress through appearance evaluation was stronger among female adolescents than among the males. Hence, while both male and female adolescents may benefit from enhanced (or maintained) bodily self-perception for reducing psychological distress, females seem to benefit more strongly. The support for the moderated mediation hypothesis fits well with the common perceptions of appearance as being particularly important to the feminine gender role and for females' evaluation of their self-worth in the Western culture. For example, research suggests that boys may be less exposed to sociocultural pressure to be thin than girls, and the drive for thinness is stronger among females than males (Knauss, Paxton & Alsaker, 2007; Ricciardelli & McCabe, 2001). Indeed, as indicated by higher appearance orientation scores among the girls in the present sample, they reported higher investment in their appearance than did boys. While Presnell, Bearman and Stice (2004) found evidence that perceived peer pressure to be thin predicted depression among adolescent girls mediated by body dissatisfaction, Siegel (2002) reported that gender differences in rates of depressive symptoms were largely eliminated when variations in body dissatisfaction was controlled for. These results fit well with our findings, and help explain the gender-specific role of appearance evaluation when it comes to gender differences shown in both anxiety and depression during adolescent years (Ohannessian et al., 1999; Seiffge-Krenke & Stemmler, 2002; Siegel, 2002).

The hypothesized model only accounted for 16% of the variation in psychological distress (R2 = .16). Although the total explained variance should be considered modest, the results from this study may well be considered meaningful. The findings are theoretically informed (Abelson, 1985; Prentice & Miller, 1992), fitting to the larger theoretical framework regarding the role of physical activity and self-esteem on mental health issues during adolescence (e.g., Sonstroem, 1997). Taken together with other factors associated with psychological distress, this study adds to this cumulative base of evidence that physical activity may play a role for aspects of young individuals' mental health. Furthermore, to paraphrase Prentice and Miller (1992), while the variance explained in psychological distress may not be regarded impressive, this should not result in reconsidering the importance of the psychological process of interest.

Limitations

Our study aimed to contribute to the understanding of how and for whom physical activity may help alleviate psychological distress in the format of depression and anxiety.

The findings obtained must be evaluated in light of study limitations. Due to the cross-sectional design, no conclusions can be drawn regarding causal relations. Longitudinal studies and well-designed controlled trials are warranted to establish causal proofs. Furthermore, physical appearance is only one of several plausible self-esteem concepts that may contribute to the understanding of the physical activity – psychological distress link. Additional mediators should be addressed in future studies.

Also, a closer inspection of potential differences between specific sports and physical activities when it comes to alleviate psychological distress would seem worthwhile. While characteristics embedded in some activity and sports may help generate constructive bodily self-perceptions in benefit of psychological health, participation in others such as those favoring leanness and a specific weight may facilitate pressure to be thin, enhance maladaptive physical self-perceptions, and onset eating pathology and depression (Sundgot-Borgen, 1994).

Moreover, even though the factors of interest in this study are hypothesized to be student-level constructs, the sample in this study is multilevel (students are nested within different schools). Even though the hypothesized model includes schools as covariates and thus taking the possible school-clustering into account, the fact that the sample is drawn at school-level and not student-level should be considered when interpreting the results of this study.

Furthermore, the MBRSQ-AS subscales were not originally validated for adolescents under the age of 15. Whether this instrument is valid for 13- and 14-year-old teenagers could therefore be questioned. We conducted separate internal consistency analyses and factor analyses for this subsample revealing internal consistency coefficients and factor loadings similar to those aged 15–18.

The present sample may be considered representative for the general population of Norwegian adolescents. However, the prevalence of serious mental health concerns or diagnosed mental illness in the sample remains unknown. This may confound the findings in this study, and should be considered when interpreting the results.

Balancing strengths and limitations, study findings add to the literature on potential mechanisms operating to explain mental health benefits from taking part in physical activity in male and female adolescents.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

A better understanding of mechanisms linking physical activity to mental health is important for the development of optimal intervention programs for young people. Our findings suggest that physical activity participation is indirectly related to lower level of psychological distress through positive evaluation of one's appearance. This indirect effect was conditional of gender, indicating that the indirect effect was stronger in female adolescents than in males. Although longitudinal studies and randomized controlled trials are needed to assess the causal direction of the relationships, this study, together with others in the field, may provide empirical justification for this. Consistent with the argument put forward by Biddle and Asare (2011), in future studies, measures of physical factors such as body-image indices should be taken into account to better understand the mechanisms of mental health outcomes of physical activity and sport for young people.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

This study was institutionally funded by the University of Agder. T.H. was the lead author of the manuscript and helped to collect the data, performed the data analysis, and drafted the manuscript.

Y.O. and B.T.J. assisted by critically reviewing and commenting on the first draft. All authors critically reviewed and approved the final version submitted for publication.

The authors have declared that they have no competing or potential conflicts of interests.

We acknowledge the contribution of Dr. Reidar Säfvenbom in generating the “Goodness of fit in Norwegian Youth Sport”-study and we appreciate the anonymous reviewer in giving valuable feedback on an earlier version of this manuscript.

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  2. Abstract
  3. Introduction
  4. Method
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References
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