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Lamprecht M, editor. Antioxidants in Sport Nutrition. Boca Raton (FL): CRC Press/Taylor & Francis; 2015.

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Chapter 13Methodological Considerations When Evaluating the Effectiveness of Dietary/Supplemental Antioxidants in Sport

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In recent years, there has been an increasing practical, clinical and scientific interest in the effects of free radicals and antioxidants related to sporting activities and exercise performance (Nikolaidis et al. 2012a,b; Peternelj and Coombes 2011; Powers et al. 2011; Reid 2008; Stear et al. 2009). Most of these reviews agree that oxidative stress represents a fundamental biological response to exercise stimuli. The generation of reactive oxygen species (ROS) increases with inclining oxygen utilisation during endurance exercises and is potentially associated with the risk of damage to muscles and other tissues. The key question is how effectively athletes can defend against the increased free radicals resulting from exercise. Do athletes need to take extra antioxidants? Studies provide arguments for and against the use of antioxidants but also emphasise that additional research will be required until a final judgement can be made (Peternelj and Coombes 2011; Powers et al. 2011; Stear et al. 2009). Obviously, large discrepancies exist between scientific evidence and the promotion by manufacturers and distributors of antioxidants. The promises of manufacturers and distributors are often based on anecdotal reports, not well-controlled observational studies or even on theoretical assumptions or speculations. It is readily understandable that the expectations of athletes encourage business interests. However, the main goal of scientific studies is to find the truth. To accomplish this goal, appropriate study design and sound statistical methods are of utmost importance.


Subjects taking nutritional supplements such as antioxidants expect ‘truly’ beneficial effects. Such an expectation may sometimes provoke substantial placebo effects which are often misinterpreted as true effects. Most of the consumers, however, will not really experience benefits. Thus, a rigorous scientific and practical verification of true effects is paramount. Based on the definition of John Dewey, a student of the pragmatic philosophers Sanders Pierce and William James, truth should be coherent with other knowledge, should be repeatable, and lead to correct predictions (Spector 2009). To meet these requirements, rigorous scientific methods have to be used, finally enabling the prediction of effects, for example, those of supplemental antioxidants.


There are three types of research or studies advancing scientific knowledge: descriptive, explanatory and predictive research.

13.3.1. Descriptive Studies

Descriptive studies, also referred to as observational studies, collect information on existing relationships, for example, between health status or level of performance and the regular intake of antioxidants. They include not only cross-sectional and longitudinal (cohort) studies but also studies on single cases and case series. Whereas cross-sectional studies look on existing associations at a single time point or time interval within a defined population of interest, longitudinal studies follow the individuals of such a population over time. These studies do not change the environment or conditions but simply describe ‘what is’ or ‘what was’. The findings obtained from these studies provide associations between outcome variables but do not allow causality to be established. Thus, after this first step of research, explanatory and predictive studies are necessary to assess causality.

13.3.2. Explanatory Studies

Explanatory studies try to answer ‘how’ and ‘why’ an intervention works. For instance, when descriptive research found associations between the intake of antioxidants and high levels of performance, it will be of interest to show ‘how’ and ‘why’ antioxidants can improve performance, that is, to establish cause and effect relationships. Interventions in explanatory studies are strictly defined and controlled. Randomised controlled trials (RCT) are considered as simple and powerful tools in clinical research. In this type of study, participants are randomly assigned to the intervention group, for example, receiving supplemental antioxidants, or to the control group, for example, receiving placebo. To avoid biases (systematic errors), it is highly desirable that researchers performing outcome measurements as well as study participants are ‘blinded’, that is, they do not know whether somebody has been allocated to the intervention or the control group.

13.3.3. Predictive Research

Predictive research forecasts intervention effects based on the knowledge gathered by descriptive and experimental studies. For example, supplemental antioxidants, which have been demonstrated to be beneficial in descriptive studies and cause and effect relationship, have also been shown in explanatory studies and are consumed by a defined population, for example, athletes. Ideally, beneficial effects of the intervention should be predictable in a large proportion of the subjects studied.


The most important reasons for prasticing evidence-based medicine (EBM) or evidence-based practice (EBP) are to improve quality of care by the use of practices that really work, and the elimination of those that do not work or are even harmful (Gray and Pinson 2003). According to Sackett et al. (1996), EBM means using the best evidence in making decisions about the care of the individual patient based on the best available evidence from systematic research integrating individual clinical expertise and patient values. The same is true with regard to medical care and support for athletes. The higher the level of evidence (Table 13.1), the better the quality of research and the more likely that the predicted effects will actually occur.

TABLE 13.1

TABLE 13.1

Levels of Evidence


When planning studies of antioxidant effects on exercise performance, a comprehensive knowledge of integrated biochemical and physiological mechanisms of exercise metabolism, free radicals and antioxidants is essential. The following section intends to provide some general thoughts and will focus on potentially negative effects of antioxidants. These adverse effects are discussed in more detail in other chapters of this book.

The dietary vitamin and supplement industry is one of the world’s fastest growing markets, with 32 billion dollars in revenue for nutritional supplements alone (www.forbes.com). Although it is generally accepted that the prevalence of vitamin deficiency is low in industrialised countries and physically active individuals do not show increased requirements of vitamins and minerals, food supplements are increasingly used by the general population and athletes. There is increasing evidence that antioxidants may not only lack health benefits but may even prevent health-promoting effects of physical exercise in humans (Ristow et al. 2009). Bjelakovic et al. (2007) looked at data from 67 studies on antioxidant supplementation and they concluded that β-carotene, vitamin A and vitamin E supplementation seemed to increase the risk of death. When a 6-week aerobic exercise training programme was carried out by patients with hypertension, supplementation of antioxidants (vitamins C and E and lipoic acid) maintained the hypertensive status of subjects and the inhibition of exercise-induced flow-mediated vasodilatation (Wray et al. 2009). Although a possible explanation (free radical acting as vasodilators) is put forward, the authors do not provide experimental evidence for the mechanism of their finding. Also, though the training period is rather short (6 weeks), the authors fail to include a time control group to account for a time effect. The authors apply spin trapping and electron paramagnetic resonance spectroscopy (EPR) to assess plasma-free radical concentration, which is considered the gold standard method. However, they used a different training modality (graded maximal cycling) than was used for the exercise training programme itself (knee-extensor exercise). Secondary, stress-related effects due to a difference in exercise modality for training and testing cannot therefore be excluded. Ristow et al. (2009) found a blunted induction of gene expression and an increase in insulin sensitivity in a vitamin C and E supplemented group undergoing about 60 min of circuit-type training 5 times a week for 4 weeks. By contrast, only the non-supplemented group increased the ROS-dependent transcription factors and insulin-mediated glucose disposal in response to the exercise training. Thiobarbituric acid-reactive substances (TBARS) levels were significantly increased in response to exercise in the non-supplemented group but not in the group taking the antioxidants. Ristow et al. (2009) emphasises the importance of oxidative stress to induce the above-mentioned changes. Plasma levels of vitamins C and E were not determined in the participants and, surprisingly, Ristow et al.’s report (2009) increased mRNA levels of PGC-1α, PGC-1ß, PPARβ as well as superoxide dismutase (SOD) 1 and 2 only in the non-supplemented group. Along these lines, it is worth mentioning that euglycemic–hyperinsulinemic clamps and muscle biopsies were performed 1 week after completion of training. It has been shown that increases in the mRNA levels of the above-mentioned transcription factors in response to exercise are transient in nature and return to baseline levels about 24 h after an exercise bout (Pilegaard et al. 2003). The same is true for training-induced increases in insulin-stimulated glucose disposal which quickly decays 3–4 days after cessation of training, being undetectable after 7 days (Oshida et al. 1991; Burstein et al. 1985). Thus, owing to some methodological flaws, the findings by Ristow et al. (2009) are difficult to comprehend. Further, the number of participants to be included to observe a true effect was not planned, which reduces the validity of the study. The authors state that ‘supplementation with antioxidants may preclude these health-promoting effects of exercise in humans’. This conclusion is not comprehensive, given the fact that hundreds of different antioxidants interplay to regulate the pro-oxidative–antioxidative balance in the body. Therefore, this statement cannot be extrapolated to antioxidants in general by using only two specific antioxidants.

Similarly, Yfanti et al. (2011) studied the effect of vitamin C and E supplementation on insulin sensitivity in response to endurance training. The training consisted of a mixture between interval and continuous cycling ~80 min, 5 times a week for 12 weeks. The authors report similar increases in insulin-stimulated glucose uptake in both groups of about 15% as well as VO2max of about 18% in both groups. They concluded that supplementation with vitamins C and E for 12 weeks of cycling exercise training had no effect on performance or insulin-stimulated glucose uptake. Unfortunately, no markers of oxidative stress were reported in this study providing no information on the effectiveness of antioxidant supplementation on levels of oxidative stress.

It is interesting that both Ristow et al. (2009) and Yfanti et al. (2011) use a similar regime and yet the outcome of their studies is distinct. A closer look, however, reveals a few differences between the two studies: Yfanti et al. (2011) provide 500 mg vitamin C and 400 IU vitamin E daily for 16 weeks (4 weeks lead-in phase), whereas in the study of Ristow et al. (2009), participants took double the amount of vitamin C (1000 mg) and the same dose of vitamin E (400 IU) daily for a total time of only 4 weeks. In the Ristow study, the supplement dosages of vitamin C and vitamin E for the participant’s age group are therefore 10 and 17 times higher, respectively, than the recommended dietary allowances. Also, the total time training is very different, being 4 weeks, 5 times a week (20 sessions) in the Ristow study and 12 weeks, 5 times a week (60 sessions) for the Yfanti study. Thus, more research is needed to clarify whether these parameters can explain the observed differences between the two studies. Furthermore, it is likely that methodological issues may partly account for the inconsistent results when comparing these two studies (see above).

ROS seem to play a role in disease prevention and the extension of the lifespan. ROS are actually ‘required’ for the extension of the lifespan, by a mechanism that is called mitohormesis (Radak et al. 2008; Ristow 2012). Antioxidants that interfere with ROS formation seem to prevent this effect (Gomez-Cabrera et al. 2012). This finding is discussed controversially (Higashida et al. 2011), but the vast majority of experimental evidence clearly advises against supplementation of antioxidative vitamins. Additionally, there is also growing evidence that antioxidative vitamins may play a role in the development of obesity (Mangge et al., 2013).

The number of overweight and obese individuals has reached epidemic proportions with serious social and psychological dimensions. For this development, life-style changes with increased consumption of more energy-dense but nutrient-poor foods with high levels of sugar and saturated fats, combined with reduced levels of physical activity, are considered to be most important. Reduced caloric intake in combination with regular physical activity is certainly the most effective way of preventing the development of obesity.

Recently, it was claimed that antioxidant supplements diminish the health-promoting effects of exercise in patients with type 2 diabetes (Ristow et al. 2009). As the most important mode of action, antioxidants neutralise ROS and thus counteract physiological responses to exercise-induced ROS. However, this relationship could be of much broader relevance and some other possible consequences could be extrapolated that are more generally relevant (Theodorou et al. 2011).

The increased mitochondrial formation of ROS induced by exercise (Powers and Jackson 2008; Kyparos et al. 2009) is central to oxidation of carbon fuels. It is also well documented that physical exercise and sport activities elicit a significant activation of inflammatory responses and create an oxidising milieu which involves the release of specific immune products like cytokines and neopterin and their serum-soluble receptors, sIL-2R, sTNF-R55 and sCD23 (Tilz et al. 1993). The pro-inflammatory cytokine cascade is activated by signal transduction elements such as nuclear factor-kappaB (NF-ϰB), which is inducible by ROS and critical for establishing a pro-inflammatory response (Schreck et al. 1991). Antioxidant compounds are able to dampen this pro-inflammatory response. As a consequence, ROS production as well as the molecular mediators of endogenous ROS defence, such as superoxide dismutase and glutathione peroxidise, are diminished in response to physical exercise (Ristow et al. 2009). However, whether this finding justifies their conclusion that antioxidant supplements prevent the health-promoting effects of physical exercise is probably too far-reaching and questionable, because the induction of antioxidant mediators cannot be regarded as the one and only benefit of sport and physical exercise.

Regular physical activity may be considered the most important condition counteracting metabolic and cardiovascular diseases (Szostak and Laurant 2011). There is ample evidence that physical activity is an independent and important factor for the assessment of cardiovascular risk and that increasing levels of physical fitness protect against elevations in most risk factors in subjects with and without cardiovascular diseases. Although, in general, there seems to be a reduction in biomarkers of oxidative stress with antioxidant intake, the physiological meaning of this is not clear and the significance for health implications is still poorly understood. The ergogenic potential of antioxidants is still debated; however, a meta-analysis of 68 randomised antioxidant supplement trials revealed that antioxidant intake does not improve overall health but may in fact increase mortality (Bjelakovic et al. 2007). Further studies are required before final conclusions can be drawn.


This section is not intended to be comprehensive but tries to provide some representative examples underpinning the necessity of careful study planning. For an extensive review, the reader may refer to Peternelj and Coombes (2011).

Many athletes take vitamins and antioxidants with the expectation of increasing exercise performance (Huang et al. 2006). Athletes seem to use supplements more frequently than the general population, with a prevalence of about 46% in athletes and an increasing prevalence with higher levels of professionalism (Sobal and Marquart 1994). Therefore, it has long been of interest to athletes and coaches as well as to scientists whether the support of the endogenous antioxidant defence systems by additional supplementation of antioxidants is an effective strategy to reduce oxidative stress and ROS production and hence to possibly improve exercise performance. However, there is evidence that ROS are important signalling molecules that act on pathways essential to and beneficial for exercise-induced adaptations (Burgoyne et al. 2007; Powers et al. 2010) with regard to tissues such as skeletal muscle. As early as 1971, it was shown that vitamin E supplementation (400 IU/day for 6 weeks) caused unfavourable effects on endurance performance in swimmers (Sharman et al. 1971), and more recent studies also suggest negative effects of antioxidant supplementation such as blunted adaptation to exercise stimuli or promotion of exercise-induced oxidative stress (Malm et al. 1996, 1997; Avery et al. 2003; Nieman et al. 2004).

The number of antioxidants presented in the scientific literature and on the market is countless, including substances such as colostrum, caffeine, selenium, carotenoids such as β-carotene, lycopene, lutein, zeaxanthin, coenzyme Q and many others. It is worth mentioning that most of these substances have not undergone proper evaluation and that there is insufficient scientific evidence regarding the ergogenic effects and long-term safety. In this section, we will therefore focus on studies assessing the connection between antioxidant supplementation and performance using either vitamin E or C as they are among the most powerful and well-characterised antioxidants (Beyer 1994). Vitamin E is one of the most important nutritional antioxidants accounting for membrane stability and fluidity by preventing lipid peroxidation (Jiang et al. 2011). Vitamin C contributes to the prevention of lipid peroxidation in interstitial fluid and plasma (Bradshaw et al. 2011). Different studies report blunted adaptations to the endurance exercise. Gomez-Cabrera et al. (2008) found a significant increase of ~186% in endurance performance in rats subjected to running for 6 weeks, 5 days/week on an animal treadmill. Endurance performance was significantly blunted (increase ~26%) in rats that received a daily dose of vitamin C (0.24 mg/cm2 body surface area). The authors found that vitamin C supplementation had a significantly negative effect on mitochondrial biogenesis. VO2max, however, was not negatively affected by antioxidant supplementation along the lines of an excess capacity of muscle mitochondria over O2 delivery by the circulation (Boushel et al. 2011). Similar results were obtained in the same study with men training for 8 weeks (Gomez-Cabrera et al. 2008). Subjects receiving an oral dose of 1 g vitamin C per day increased VO2max by 11% compared to a 22% increase in the non-supplemented group after 8 weeks of training. The results were not significant, probably due to a small sample size of only five people in the supplemented group. Also, no mechanistic pathway analysis from muscle tissue obtained from biopsies was performed in the human group due to ethical restraints. Although it is, in general, commendable to include a human and an animal trial in one study, direct comparisons between humans and rats cannot be made and translations of results are not always straightforward. It is worth mentioning that inbred rats are nearly genetically identical compared to human subjects, who are inherently genetically diverse. Sample size calculations based on findings from homogeneous, nearly identical animals would therefore lead to an underestimation of subjects in human studies. This study represents a typical example where findings from an animal study were not confirmed in the human study branch possibly due to a type 2 error. The use of an adequate sample size is a prerequisite to obtaining meaningful results.

In another human study by Khassaf et al. (2003) the negative effects of ascorbic acid supplementation on the adaptive responses of endogenous antioxidant enzymes and stress proteins were demonstrated. The authors found increased baseline activities of SOD, catalase (CAT) and HSP60 on vitamin C supplementation, which could be an indication of a pro-oxidant effect of vitamin C (Carr and Frei 1999). However, it still remains unclear whether this up-regulation of defence mechanisms is beneficial or deleterious to the tissue. It is also not clear whether suppression of the expression of these proteins following stress to skeletal muscle will be beneficial to skeletal muscle viability over the longer term. Further studies are necessary to elaborate on these questions. Furthermore, it has been shown that supplementation with ascorbic acid does not preserve muscle function but hinders the recovery process, thereby being detrimental to future performance (Close et al. 2006).

Asha Devi et al. (2003a, 2003b) studied the effect of vitamin E supplementation on swimming performance in rats of different age groups (4, 8, 12 and 22 months of age). The authors report significant differences in endurance capacity between supplemented and control rats over all age groups studied with vitamin E-supplemented rats showing higher endurance capacity. Further, supplementation with vitamin E resulted in a beneficial plasma lipid profile. The findings of Asha Devi et al. suggest an ergogenic effect of vitamin E supplementation on exercise performance. However, not all animal studies have shown performance increases following antioxidant administration. As an example, rats supplemented with vitamin E failed to improve treadmill time to exhaustion in two studies (Mehlhorn et al. 1989; De Oliveira et al. 2003). Exercise per se lead to an up-regulation of CAT and SOD activity in all age groups except the old group. Old animals only showed increased SOD activity when supplemented with vitamin E. This might indicate that supplementation is not necessary during youth. Malondialdehyde (MDA) content was lower in supplemented rats compared to the non-supplemented control group. Interestingly, the authors report reduced tissue vitamin E levels in the control group concluding that the antioxidant is utilised during exercise to scavenge free radicals. Importantly, this is again just true for the animals from the old group. As for the aforementioned studies also in this study, the sample size is rather low and it remains elusive whether these findings are transferable to humans. Whereas a number of studies report an ergogenic effect of antioxidants, the majority of studies found no changes or even negative outcomes. For a review, see Peternelj and Coombes (2011).

Intake of dietary supplements is very prevalent among the community of professional athletes (Huang et al. 2006). It is well established that a number of commercially available dietary supplements can be cross-contaminated with prohibited substances found on the Prohibited List of the World Anti-Doping Agency (Geyer et al. 2008). The intake of dietary supplements is therefore not only connected with a high risk of inadvertent doping but can also jeopardise the health of an athlete. Athletes should therefore use caution when considering supplementation with antioxidants, especially at higher doses.

In one particular case, the use of antioxidants for athletes may be justified. Dietary recall studies show that some athletes do not consume a balanced diet containing enough fruits and vegetables, and therefore these athletes potentially lack antioxidants (Farajian et al. 2004). In this case, it may be necessary to supplement with antioxidants in reasonable dosages to achieve physiological levels of antioxidants. Apart from that, athletes are rather encouraged to focus on a healthy, energetically adequate diet that is rich in antioxidant-containing foods (such as whole grains, fruits, vegetables, nuts and seeds). A balanced diet is the best nutritional approach to optimise a person’s antioxidant status.


The studies mentioned above show inconclusive results regarding the effects of antioxidants on health benefits and exercise performance. One limitation of these studies is the training status of the subjects. Participants are mostly untrained or moderately trained subjects with a VO2max in the range of around 45–60 mL/kg/min. Besides a limited number of recreationally active individuals who take supplements, it is mostly highly trained or even professional athletes who use antioxidants. It appears that there is a considerable difference with regard to oxidative stress between a single bout and regular physical activity. Repeated bouts of physical activity such as are experienced by athletes induce an up-regulation of the endogenous antioxidant defence system to minimise oxidative damage (Radak et al. 2008), whereas a single bout of exercise seems to be insufficient to induce these adaptations (Ji 2008). It is therefore not surprising that a number of markers of oxidative stress, for example, protein carbonyls (PC) and MDA appear to be unaffected in trained subjects in response to normal or even strenuous exercise (Farney et al. 2012; Bloomer et al. 2006) or if at all, seem to be more affected by a high fat diet than by exercise (McCarthy et al. 2013). These data suggest that oxidative stress is not elevated in exercise-trained subjects. Further, most of the studies only include an insufficient number of subjects and can therefore be considered as underpowered. Most studies do not provide sample size or power calculations in order for the reader to comprehend the likelihood that the findings are real. An important addition in every study would be to characterise the endogenous redox state of the subjects at baseline to characterise their defence system. It can be speculated that the endogenous defence system is better developed in well-trained than in untrained people, which again has an influence on the outcome in these different populations. Factors such as training status, age and sex have not been extensively studied and may have a considerable impact on oxidative stress response to exercise. Lastly, the site of occurrence between different organs and timing of the maximal effect of oxidative stress in response to an exercise bout have to be considered.

For future directions, it will be important for researchers to incorporate other substances than vitamins C, E and coenzyme Q10 based on the large number of available antioxidants that warrant systematic testing. The fact that these three substances were mainly studied makes generalisations difficult.

Regarding study design, a crossover design would be a clean way to study the effects of antioxidants. Also, established animal models of increased or decreased oxidative stress (e.g. the superoxide dismutase 2 nullizygous mouse as a model for increased oxidative stress and the transgenic mouse over-expressing mitochondrial catalase such as the mCAT mouse model for decreased oxidative stress) may prove very useful as positive or negative controls. It is, however, important to mention that findings from animal experiments cannot be automatically translated to humans.

Plasma volume decreases due to exercise-induced dehydration. This fact might be responsible for an overestimation of post-exercise markers of oxidative stress in studies that did not account for the changes in plasma volume.

A general problem of in vivo detection of antioxidants in humans lies in their short-lived nature. Although antioxidants have been detected ex vivo (Ashton et al. 1998), the transition to their behaviour in vivo is therefore often not necessarily scientifically sound. Free radicals exhibit a high reactivity and a relatively short half life (e.g. 10−5s for superoxide radicals and 10−9s for hydroxyl radical) (Fisher-Wellman and Bloomer 2009). They are therefore difficult to measure directly. A direct measurement method and gold standard of free radical assessment is electron spin resonance spectroscopy using spin traps (Ashton et al. 1998). Although these methods provide accurate information their application is costly and requires specialist personnel. That is why the majority of researchers make use of indirect methods that have been developed to assess biomarkers of oxidative stress. These markers are always selective and never thorough, and studies using a variety of markers do not always provide consistent results (Vollaard et al. 2005). As a general rule, it can be stated that the more markers that are assessed the better. Investigating only one marker does not give a representative view of the oxidative stress status of the subject. One extensively used marker is TBARS, a reagent commonly used to assay MDA, which is an end product of lipid peroxidation. As there are other cellular sources of MDA, the TBARS assay is not entirely representative for lipid peroxidation (Trevisan et al. 2001). The fact that TBARS is not specific for MDA is often ignored (Bird and Draper 1984). Methodological shortcomings such as TBARS production during the assay (Vollaard et al. 2005) have to be taken into account and different markers of oxidative stress (e.g. isoprostane, lipid hydroperoxide, conjugated dienes and pentane) have to be considered. To account for methodological problems of assessing biomarkers of oxidative stress, it is therefore paramount to include a non-exercising control group as the baseline readout. Furthermore, information on exact procedures of assays is often neglected but should be included in every publication to comprehend the outcome. Similar methodological constraints apply when assessing levels of the popular antioxidant marker glutathione with its reduced (GSH) and oxidised (GSSG) fractions.


Proper planning of the study is essential. A simple checklist is provided in Table 13.2.

TABLE 13.2

TABLE 13.2

Checklist to Support Study Planning

13.8.1. Rationale, Study Aim and Hypothesis

Numerous studies have investigated the effects of antioxidant supplementation on exercise performance, each having strengths but to some extent also weaknesses (Simon-Schnass and Pabst 1988; Reid et al. 1994; Matuszczak et al. 2005; Subudhi et al. 2006; Matzi et al. 2007; Gatterer et al. 2013). The rationale to study such effects is the hypothesis that oxidative stress possibly contributes to muscle fatigue and might impair exercise performance (Reid 2001). Supposed mechanisms are impairments of muscle and cellular function due to the modifications or damages of contractile proteins, suppression of the calcium sensitivity of myofilaments, alterations of mitochondrial function and its degradation (Callahan et al. 2001; Ott et al. 2007; Murphy et al. 2008). Consequently, eliminating oxidative stress is assumed to improve performance.

When investigating within this field, knowledge of the specific literature is of utmost importance. This enables gaps to be established in the research and is valuable in formulating the study rationale and the study aims. Importantly, any interventional study greatly benefits from a prospective hypothesis and a study design that makes it possible to accept or reject the hypothesis within the primary end point (Preiser et al. 2002). Within the field of nutritional studies, a double-blinded, randomised and placebo-controlled study design is favoured.

13.8.2. The Study Participant

When selecting participants it is important to recognise that various characteristics of the participants might influence the outcome of the investigation. These include anthropometric and demographic parameters (e.g. body composition, age and sex distribution), type of practised sport (e.g. cycling vs. running), fitness level (e.g. recreationally active individuals vs. elite athletes), training periodisation (e.g. off-season vs. in-season) and baseline oxidative stress and defence variables (e.g. participants with high vs. low levels of oxidative stress at baseline). Thus, selection depends on the study aims and hypotheses. Moreover, chronic and/or acute diseases of the participants and behavioural factors, including smoking, caffeine and alcohol consumption, drug use and special diet, need to be controlled and might, according to the study goal, be considered as exclusion criteria. It has to be mentioned that studying healthy trained subjects having a balanced oxidative and antioxidative status already at the start of the investigation might challenge the detection of effects of a supplementation (Ellinger et al. 2011).

13.8.3. Randomisation and Blinding

After the selection of participants, a random assignment to different groups (intervention or placebo) is advisable. Stratification for fitness level, oxidative status, age and sex results in homogeneous groups, and allows documentation of possible intervention effects. It is important that participants as well as investigators involved in data processing are, at best, blinded to the intervention. Needless to say, participants should receive antioxidants or placebos that are identical in taste and appearance, and only investigators not involved in data processing are allowed to manage group allocation and supplement distribution. This procedure guarantees that placebo effects and investigator biases can be excluded.

13.8.4. Sample Size and Statistical Power

To obtain appropriate results, the use of an adequate sample size is a prerequisite. Established statistical procedures will help to choose proper sample sizes. It is not enough to reject the null hypothesis only because of statistical significance, but also because of practical importance and clinical relevance. Further, type I and type II errors have to be considered. A type I error (corresponding to a significance level of usually 0.05) is an incorrect rejection of the true null hypothesis. A type II or beta error is an error not rejecting a false null hypothesis. The statistical power (1-beta) is the probability that a test will reject the null hypothesis, although it is false. Thus, a beta error of 0.1 corresponds to a power of 90%, meaning a 90% probability of rejecting an actually false null hypothesis. A statistical power of at least 80% is necessary for a test to detect an effect if it actually exists.

13.8.5. Main Outcome Measures

The effectiveness of the supplementation is evaluated by measuring exercise performance and oxidative status before and after the intervention. Tests need to fit the aims of the study. With respect to exercise performance, laboratory or field tests are applicable. Within the laboratory setting, incremental exercise tests, time-to-fatigue tests or time trial tests in combination with gas analyses, lactate diagnostics and so on may provide valuable information on the physiological responses and the mechanisms leading to performance changes. Field tests are considered to resemble competition situations more closely but might miss explanatory measures. It has to be added that effects of antioxidant administration on performance may critically depend on the type of exercise testing (Reid et al. 1994). With respect to the measured oxidative stress and defence parameters, a large number of markers are investigated. Selecting parameters supposedly involved in muscle and cellular function might yield additional information. Most frequently, hydrogen peroxide, total and oxidised glutathione, 8-hydroxydeoxyguanosine, MDA, protein carbonyl content, 4-hydroxynonenal and nitrotyrosine were investigated (Subudhi et al. 2006; Nikolaidis et al. 2012a; Gatterer et al. 2013). Moreover, activity levels of CAT, CuZnSOD, MnSOD, glutathione peroxidase and plasma concentrations of α-tocopherol and β-carotene were also used to reflect antioxidative status (Subudhi et al. 2006; Nikolaidis et al. 2012a). It has to be recognised that oxidative stress markers measured in blood plasma might not reflect the conditions within the working muscles (Subudhi et al. 2006).

13.8.6. Considerations during the Experiment

The study period (i.e. long-term vs. short-term supplementation) needs to be in accordance with the aims of the study. During the supplementation period, two different procedures can be followed. First, the training practice of participants is kept unaltered, with groups performing approximately the same training load. Second, participants of both groups perform the same training load which is different from their usual training regime, for example, high altitude training or high intensity training. Such procedures allow investigation of the effects of the supplementation on specific training adaptations. In any case, a training logbook helps to document training activities. Additionally, it is advisable to maintain normal nutritional habits throughout the study and to document any changes. Participants suffering from acute infections or injuries during the intervention period preferentially have to be excluded from the study, since such conditions might impact outcomes (Schwarz 1996; Tiidus 1998).

13.8.7. Use of Appropriate Statistical Tests

Nutritional studies typically compare the efficacy of a new preparation with another and/or a placebo. Besides a pure description, it is important to know whether observed differences between groups (preparations) are just random or are really true. Inferential statistical tests commonly used in the nutrition profession have been recently summarised by Saracino et al. (2013) and these authors also report how to choose them.


Making decisions about medical care and support of athletes, including the use of dietary/supplemental antioxidants in sport, should be based on the best available evidence from systematic research. However, large discrepancies exist between scientific evidence and the promotion by manufacturers and distributors of antioxidants. When evaluating the effectiveness of dietary/supplemental antioxidants, comprehensive knowledge of integrated biochemical and physiological mechanisms of exercise metabolism, free radicals and antioxidants is as necessary as the consideration of stringent methodological standards.


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