Erythrocyte metabolism.

Our aim is to present an updated overview of the erythrocyte metabolism highlighting its richness and complexity. We have manually collected and connected the available biochemical pathways and integrated them into a functional metabolic map. The focus of this map is on the main biochemical pathways consisting of glycolysis, the pentose phosphate pathway, redox metabolism, oxygen metabolism, purine/nucleoside metabolism, and membrane transport. Other recently emerging pathways are also curated, like the methionine salvage pathway, the glyoxalase system, carnitine metabolism, and the lands cycle, as well as remnants of the carboxylic acid metabolism. An additional goal of this review is to present the dynamics of erythrocyte metabolism, providing key numbers used to perform basic quantitative analyses. By synthesizing experimental and computational data, we conclude that glycolysis, pentose phosphate pathway, and redox metabolism are the foundations of erythrocyte metabolism. Additionally, the erythrocyte can sense oxygen levels and oxidative stress adjusting its mechanics, metabolism, and function. In conclusion, fine-tuning of erythrocyte metabolism controls one of the most important biological processes, that is, oxygen loading, transport, and delivery.

Inter-individual variability in redox and performance responses after antioxidant supplementation: A randomized double blind crossover study.

AIM: We aimed to investigate the inter-individual variability in redox and physiological responses of antioxidant-deficient subjects after antioxidant supplementation. METHODS: Two hundred individuals were sorted by plasma vitamin C levels. A low vitamin C group (n = 22) and a control group (n = 22) were compared in terms of oxidative stress and performance. Subsequently, the low vitamin C group received for 30 days vitamin C (1 g) or placebo, in randomized, double-blind, crossover fashion, and the effects were examined through a mixed-effects model, while individual responses were calculated. RESULTS: The low vitamin C group exhibited lower vitamin C (-25 µmol/L; 95%CI[-31.7, -18.3]; p < 0.001), higher F2 -isoprostanes (+17.1 pg/mL; 95%CI[6.5, 27.7]; p = 0.002), impaired VO2max (-8.2 mL/kg/min; 95%CI[-12.8, -3.6]; p < 0.001) and lower isometric peak torque (-41.5 Nm; 95%CI[-61.8, -21.2]; p < 0.001) compared to the control group. Regarding antioxidant supplementation, a significant treatment effect was found in vitamin C (+11.6 µmol/L; 95%CI[6.8, 17.1], p < 0.001), F2 -isoprostanes (-13.7 pg/mL; 95%CI[-18.9, -8.4], p < 0.001), VO2max (+5.4 mL/kg/min; 95%CI[2.7, 8.2], p = 0.001) and isometric peak torque (+18.7; 95%CI[11.8, 25.7 Nm], p < 0.001). The standard deviation for individual responses (SDir) was greater than the smallest worthwhile change (SWC) for all variables indicating meaningful inter-individual variability. When a minimal clinically important difference (MCID) was set, inter-individual variability remained for VO2max , but not for isometric peak torque. CONCLUSION: The proportion of response was generally high after supplementation (82.9%-95.3%); however, a few participants did not benefit from the treatment. This underlines the potential need for personalized nutritional interventions in an exercise physiology context.

Free radicals and antioxidants: appealing to magic.

In biology, there are no good or evil molecules. There is limited or no evidence to support the consumption of antioxidants or (super)foods rich in antioxidants, for the intended purpose of an antioxidant effect, because there is risk of interfering with free radicals and deoptimizing the regulation of fundamental processes.

Skeletal muscle and erythrocyte redox status is associated with dietary cysteine intake and physical fitness in healthy young physically active men.

PURPOSE: To investigate the association between redox status in erythrocytes and skeletal muscle with dietary nutrient intake and markers of physical fitness and habitual physical activity (PA). METHODS: Forty-five young physically active men were assessed for body composition, dietary nutrient intake, muscle strength, cardiorespiratory capacity and habitual PA. Blood and muscle samples were collected to estimate selected redox biomarkers. Partial correlation analysis was used to evaluate the independent relationship of each factor with redox biomarkers. RESULTS: Dietary cysteine intake was positively correlated (p < 0.001) with both erythrocyte (r = 0.697) and muscle GSH (0.654, p < 0.001), erythrocyte reduced/oxidized glutathione ratio (GSH/GSSG) (r = 0.530, p = 0.001) and glutathione reductase (GR) activity (r = 0.352, p = 0.030) and inversely correlated with erythrocyte protein carbonyls (PC) levels (r = - 0.325; p = 0.046). Knee extensors eccentric peak torque was positively correlated with GR activity (r = 0.355; p = 0.031) while, one-repetition maximum in back squat exercise was positively correlated with erythrocyte GSH/GSSG ratio (r = 0.401; p = 0.014) and inversely correlated with erythrocyte GSSG and PC (r = - 0.441, p = 0.006; r = - 0.413, p = 0.011 respectively). Glutathione peroxidase (GPx) activity was positively correlated with step count (r = 0.520; p < 0.001), light (r = 0.406; p = 0.008), moderate (r = 0.417; p = 0.006), moderate-to-vigorous (r = 0.475; p = 0.001), vigorous (r = 0.352; p = 0.022) and very vigorous (r = 0.326; p = 0.035) PA. Muscle GSSG inversely correlated with light PA (r = - 0.353; p = 0.022). CONCLUSION: These results indicate that dietary cysteine intake may be a critical element for the regulation of glutathione metabolism and redox status in two different tissues pinpointing the independent significance of cysteine for optimal redox regulation. Musculoskeletal fitness and PA levels may be predictors of skeletal muscle, but not erythrocyte, antioxidant capacity. TRIAL REGISTRATION: Registry: ClinicalTrials.gov, identifier: NCT03711838, date of registration: October 19, 2018.

Skeletal muscle and cerebral oxygenation levels during and after submaximal concentric and eccentric isokinetic exercise.

The aim was to investigate the potential differences in muscle (vastus lateralis) and cerebral (prefrontal cortex) oxygenation levels as well as in the number of repetitions and total work output between isokinetic eccentric and concentric exercise at a moderate relative intensity until exhaustion. Ten recreationally active young men underwent two isokinetic exercise sessions either concentric or eccentric, one on each randomly selected leg. The protocols were performed at 60°/s and an intensity corresponding to 60% of the maximal voluntary contraction (MVC) of each contraction type. Concentric torque was significantly lower compared to eccentric torque in both peak values and at values corresponding to 60% of MVC [230 ± 18 Nm vs. 276 ± 19 Nm (P = .014) and 137 ± 12 Nm vs. 168 ± 11 Nm, respectively (P = .010)]. The participants performed 40% more contractions during eccentric compared to concentric exercise [122 ± 15 vs. 78 ± 7, respectively]. No differences were found in the levels of oxyhaemoglobin, deoxyhemoglobin, total haemoglobin and tissue saturation index when eccentric and eccentric exercise regimes were compared (all P > .05). Our results demonstrate that eccentric exercise of moderate intensity leads to greater resistance to fatigue and more work output compared to concentric exercise, despite the comparable muscle and cerebral oxygenation levels.

The redox signal: A physiological perspective.

A signal in biology is any kind of coded message sent from one place in an organism to another place. Biology is rich in claims that reactive oxygen and nitrogen species transmit signals. Therefore, we define a "redox signal as an increase/decrease in the level of reactive species". First, as in most biology disciplines, to analyze a redox signal you need first to deconstruct it. The essential components that constitute a redox signal and should be characterized are: (i) the reactivity of the specific reactive species, (ii) the magnitude of change, (iii) the temporal pattern of change, and (iv) the antioxidant condition. Second, to be able to translate the physiological fate of a redox signal you need to apply novel and bioplausible methodological strategies. Important considerations that should be taken into account when designing an experiment is to (i) assure that redox and physiological measurements are at the same or similar level of biological organization and (ii) focus on molecules that are at the highest level of the redox hierarchy. Third, to reconstruct the redox signal and make sense of the chaotic nature of redox processes, it is essential to apply mathematical and computational modeling. The aim of the present study was to collectively present, for the first time, those elements that essentially affect the redox signal as well as to emphasize that the deconstructing, decoding and reconstructing of a redox signal should be acknowledged as central to design better studies and to advance our understanding on its physiological effects.

The Effects of High-Intensity Interval Exercise on Skeletal Muscle and Cerebral Oxygenation during Cycling and Isokinetic Concentric and Eccentric Exercise.

The aim of the present study was to study the effects of cycling and pure concentric and pure eccentric high-intensity interval exercise (HIIE) on skeletal muscle (i.e., vastus lateralis) and cerebral oxygenation. Twelve healthy males (n = 12, age 26 ± 1 yr, body mass 78 ± 2 kg, height 176 ± 2 cm, body fat 17 ± 1% of body mass) performed, in a random order, cycling exercise and isokinetic concentric and eccentric exercise. The isokinetic exercises were performed on each randomly selected leg. The muscle and the cerebral oxygenation were assessed by measuring oxyhemoglobin, deoxyhemoglobin, total hemoglobin, and tissue saturation index. During the cycling exercise, participants performed seven sets of seven seconds maximal intensity using a load equal to 7.5% of their body mass while, during isokinetic concentric and eccentric exercise, they were performed seven sets of five maximal muscle contractions. In all conditions, a 15 s rest was adopted between sets. The cycling HIIE caused greater fatigue (i.e., greater decline in fatigue index) compared to pure concentric and pure eccentric isokinetic exercise. Muscle oxygenation was significantly reduced during HIIE in the three exercise modes, with no difference between them. Cerebral oxygenation was affected only marginally during cycling exercise, while no difference was observed between conditions. It is concluded that a greater volume of either concentric or eccentric isokinetic maximal intensity exercise is needed to cause exhaustion which, in turn, may cause greater alterations in skeletal muscle and cerebral oxygenation.

Reactive oxygen species in exercise and insulin resistance: Working towards personalized antioxidant treatment.

Reactive oxygen species (ROS) are well known for their role in insulin resistance and the development of cardiometabolic disease including type 2 diabetes mellitus (T2D). Conversely, evidence supports the notion that ROS are a necessary component for glucose cell transport and adaptation to physiological stress including exercise and muscle contraction. Although genetic rodent models and cell culture studies indicate antioxidant treatment to be an effective strategy for targeting ROS to promote health, human findings are largely inconsistent. In this review we discuss human research that has investigated antioxidant treatment and glycemic control in the context of health (healthy individuals and during exercise) and disease (insulin resistance and T2D). We have identified key factors that are likely to influence the effectiveness of antioxidant treatment: 1) the context of treatment including whether oxidative distress or eustress is present (e.g., hyperglycemia/lipidaemia or during exercise and muscle contraction); 2) whether specific endogenous antioxidant deficiencies are identified (redox screening); 3) whether antioxidant treatment is specifically designed to target and restore identified deficiencies (antioxidant specificity); 4) and the bioavailability and bioactivity of the antioxidant which are influenced by treatment dose, duration, and method of administration. The majority of human research has failed to account for these factors, limiting their ability to robustly test the effectiveness of antioxidants for health promotion and disease prevention. We propose that a modern "redox screening" and "personalized antioxidant treatment" approach is required to robustly explore redox regulation of human physiology and to elicit more effective antioxidant treatment in humans.

Priming exercise increases Wingate cycling peak power output.

PURPOSE: The aim of the present study was to investigate the effect of priming exercise on Wingate performance and fatigue. METHODS: Twelve recreationally active young male volunteers participated in the study (age: 25 ± 5 years; weight: 75.0 ± 7.5 kg; height: 177 ± 6 cm; BMI: 24.0 ± 1.7). During a first visit, participants performed a typical V˙O2max test and a supramaximal assessment of V˙O2max on a cycle ergometer, while during the next three visits, the participants performed in a random order a Wingate test (i) with no priming exercise, (ii) after priming exercise followed by a 15-min recovery (Priming15) and (iii) after priming exercise followed by a 30-min recovery (Priming30). Priming exercise lasted 6 min, at work rate corresponding to the gas exchange threshold (GET) plus 70% of the difference between the GET and V˙O2max. RESULTS: The Priming 30 condition exhibited greater peak power output (595 ± 84 W) compared to the control (567 ± 85 W) and the Priming15 condition (569 ± 95 W) (P < .05). Regarding fatigue index, a tendency towards increased resistance to fatigue was observed in the Priming30 condition compared to the control and the Priming15 conditions (P = .072). Pre-Wingate lactate levels were found to be significantly different between the Priming15 (7.18 ± 3.09 mmol/L) and the Priming30 (4.87 ± 2.11 mmol/L) conditions (P < .05). CONCLUSIONS: Priming exercise of high intensity followed by a prolonged recovery leads to increased peak power in a subsequent Wingate test. Moreover, our data are consistent with the idea that a priming exercise-induced modest increase in blood lactate concentration at the onset of the following criterion bout is a key factor of performance.

Dietary Cysteine Intake is Associated with Blood Glutathione Levels and Isometric Strength.

Glutathione is the most abundant cellular antioxidant and regulates redox homeostasis. Healthy individuals with certain antioxidant inadequacies/deficiencies exhibit impairments in physiological functions. The aim was to investigate whether low levels of dietary cysteine intake are associated with a) lower erythrocyte glutathione, b) increased plasma F2-isoprostanes, and c) impaired muscle function. Towards this aim, we recorded the dietary intake of the three amino acids that synthesize glutathione (i. e., glutamic acid, cysteine, and glycine) in forty-one healthy individuals, and subsequently measured erythrocyte glutathione levels. Maximal isometric strength and fatigue index were also assessed using an electronic handgrip dynamometer. Our findings indicate that dietary cysteine intake was positively correlated with glutathione levels (r=0.765, p<0.001). In addition, glutathione levels were negatively correlated with F2-isoprostanes (r=- 0.311, p=0.048). An interesting finding was that glutathione levels and cysteine intake were positively correlated with maximal handgrip strength (r=0.416, p=0.007 and r=0.343, p=0.028, respectively). In conclusion, glutathione concentration is associated with cysteine intake, while adequate cysteine levels were important for optimal redox status and muscle function. This highlights the importance of proper nutritional intake and biochemical screening with the goal of personalized nutrition.

Eccentric exercise per se does not affect muscle damage biomarkers: early and late phase adaptations.

PURPOSE: Acute high-intensity unaccustomed eccentric exercise performed by naive subjects is accompanied by disturbances in muscle damage biomarkers. The aim of the study was to investigate whether a causal relationship indeed exists between eccentric exercise and muscle damage. METHODS: Twenty-four men randomly assigned into a concentric only or an eccentric-only training group and performed 10 weeks of isokinetic resistance exercise (one session/week of 75 maximal knee extensors actions). Physiological markers of muscle function and damage (i.e., range of motion, delayed onset muscle soreness, isometric, concentric and eccentric peak torque) were assessed prior to and 1-3 and 5 days post each session. Biochemical markers of muscle damage (creatine kinase) and inflammation (C-reactive protein) were measured prior and 2 days post each session. RESULTS: After the first bout, eccentric exercise induced greater muscle damage compared to concentric exercise; however, during the nine following sessions, this effect progressively diminished, while after the 10th week of training, no alterations in muscle damage biomarkers were observed after either exercise protocol. Additionally, strength gains at the end of the training period were comparable between the two groups and were mode-specific. CONCLUSION: (1) eccentric exercise per se does not affect muscle damage biomarkers; (2) muscle damage occurs as a result of muscle unaccustomedness to this action type; (3) exercise-induced muscle damage is not a prerequisite for increased muscle strength. Collectively, we believe that muscle unaccustomedness to high-intensity eccentric exercise, and not eccentric exercise per se, is the trigger for muscle damage as indicated by muscle damage biomarkers.

Antioxidant supplementation, redox deficiencies and exercise performance: A falsification design.

The aim of the present study was to validate the idea of personalized redox supplementation by subjecting individuals to targeted and non-targeted antioxidant supplementation schemes. Seventy-three volunteers were screened for plasma vitamin C and erythrocyte glutathione levels. Three groups were formed: i) the "low vitamin C″ group (12 individuals with the lowest vitamin C levels; Low VitC), ii) the "low glutathione" group (12 individuals with the lowest glutathione levels; Low GSH) and iii) a control group (12 individuals with moderate vitamin C and glutathione levels). The three groups received 1 g of vitamin C or 1.2 g of NAC daily for 30 days in a crossover design with a wash-out period of 30 days. Both antioxidant treatments reduced the increased resting systemic oxidative stress levels, assessed via urine F2-isoprostanes, in the Low VitC and Low GSH groups (P < .05). A significant group × time interaction (P < .05) was found for VO2max and isometric peak torque after both treatments, with the Low VitC and Low GSH groups exhibiting improved performance only after the targeted treatment (vitamin C and NAC, respectively). A significant group × time interaction (P < .05) was found for fatigue index after NAC treatment, but not after vitamin C treatment. No interaction was found for the Wingate test after both treatments. Most of the evidence verifies the idea that antioxidant supplementation increases performance when a particular deficiency is reversed. This indicates that the presence of oxidative stress per se does not rationalize the use of antioxidants and emphasizes the need to identify "responsive" phenotypes.

Quantitative Redox Biology of Exercise.

Biology is rich in claims that reactive oxygen and nitrogen species are involved in every biological process and disease. However, many quantitative aspects of redox biology remain elusive. The important quantitative parameters you need to address the feasibility of redox reactions in vivo are: rate of formation and consumption of a reactive oxygen and nitrogen species, half-life, diffusibility and membrane permeability. In the first part, we explain the basic chemical kinetics concepts and algebraic equations required to perform "street fighting" quantitative analysis. In the second part, we provide key numbers to help thinking about sizes, concentrations, rates and other important quantities that describe the major oxidants (superoxide, hydrogen peroxide, nitric oxide) and antioxidants (vitamin C, vitamin E, glutathione). In the third part, we present the quantitative effect of exercise on superoxide, hydrogen peroxide and nitric oxide concentration in mitochondria and whole muscle and calculate how much hydrogen peroxide concentration needs to increase to transduce signalling. By taking into consideration the quantitative aspects of redox biology we can: i) refine the broad understanding of this research area, ii) design better future studies and facilitate comparisons among studies, and iii) define more efficiently the "borders" between cellular signaling and stress.

Chronic administration of plasma from exercised rats to sedentary rats does not induce redox and metabolic adaptations.

The present study aimed to investigate whether endurance exercise-induced changes in blood plasma composition may lead to adaptations in erythrocytes, skeletal muscle and liver. Forty sedentary rats were randomly distributed into two groups: a group that was injected with pooled plasma from rats that swam until exhaustion and a group that was injected with the pooled plasma from resting rats (intravenous administration at a dose of 2 mL/kg body weight for 21 days). Total antioxidant capacity, malondialdehyde and protein carbonyls were higher in the plasma collected from the exercised rats compared to the plasma from the resting rats. Νo significant difference was found in blood and tissue redox biomarkers and in tissue metabolic markers between rats that received the "exercised" or the "non-exercised" plasma (P > 0.05). Our results demonstrate that plasma injections from exercised rats to sedentary rats do not induce redox or metabolic adaptations in erythrocytes, skeletal muscle and liver.

DNA Damage Following Acute Aerobic Exercise: A Systematic Review and Meta-analysis.

BACKGROUND: Exercise is widely recognised for its health enhancing benefits. Despite this, an overproduction of reactive oxygen and nitrogen species (RONS), outstripping antioxidant defence mechanisms, can lead to a state of (chronic) oxidative stress. DNA is a vulnerable target of RONS attack and, if left unrepaired, DNA damage may cause genetic instability. OBJECTIVE: This meta-analysis aimed to systematically investigate and assess the overall effect of studies reporting DNA damage following acute aerobic exercise. METHODS: Web of Science, PubMed, MEDLINE, EMBASE, and Scopus were searched until April 2019. Outcomes included (1) multiple time-points (TPs) of measuring DNA damage post-exercise, (2) two different quantification methods (comet assay and 8-oxo-2'-deoxyguanosine; 8-OHdG), and (3) protocols of high intensity (≥ 75% of maximum rate of oxygen consumption; VO2-max) and long distance (≥ 42 km). RESULTS: Literature search identified 4316 non-duplicate records of which 35 studies were included in the meta-analysis. The evidence was strong, showcasing an increase in DNA damage immediately following acute aerobic exercise with a large-effect size at TP 0 (0 h) (SMD = 0.875; 95% CI 0.5, 1.25; p < 0.05). When comparing between comet assay and 8-OHdG at TP 0, a significant difference was observed only when using the comet assay. Finally, when isolating protocols of long-distance and high-intensity exercise, increased DNA damage was only observed in the latter. (SMD = 0.48; 95% CI - 0.16, 1.03; p = 0.15 and SMD = 1.18; 95% CI 0.71, 1.65; p < 0.05 respectively). CONCLUSIONS: A substantial increase in DNA damage occurs immediately following acute aerobic exercise. This increase remains significant between 2 h and 1 day, but not within 5-28 days post-exercise. Such an increase was not observed in protocols of a long-distance. The relationship between exercise and DNA damage may be explained through the hormesis theory, which is somewhat one-dimensional, and thus limited. The hormesis theory describes how exercise modulates any advantageous or harmful effects mediated through RONS, by increasing DNA oxidation between the two end-points of the curve: physical inactivity and overtraining. We propose a more intricate approach to explain this relationship: a multi-dimensional model, to develop a better understanding of the complexity of the relationship between DNA integrity and exercise.

Acute nicotinamide riboside supplementation improves redox homeostasis and exercise performance in old individuals: a double-blind cross-over study.

PURPOSE: Older individuals suffer from low NADH levels. We have previously shown that nicotinamide riboside [NR; a NAD(P)(H) precursor] administration impaired exercise performance in young rats. It has been suggested that supplementation of redox agents exerts ergogenic effect only in deficient individuals. We hypothesized that old individuals would more likely benefit from NR supplementation. We investigated the effect of acute NR supplementation on redox homeostasis and physical performance in young and old individuals. METHODS: Twelve young and twelve old men received NR or placebo in a double-blind cross-over design. Before and 2 h after NR or placebo supplementation, blood and urine samples were collected, while physical performance (VO2max, muscle strength, and fatigue) was assessed after the second blood sample collection. RESULTS: At rest, old individuals exhibited lower erythrocyte NAD(P)H levels, higher urine F2-isoprostanes and lower erythrocyte glutathione levels compared to young (P < 0.05). NR supplementation increased NADH (51% young; 59% old) and NADPH (32% young; 38% old) levels in both groups (P < 0.05), decreased F2-isoprostanes by 18% (P < 0.05), and tended to increase glutathione (P = 0.078) only in the old. NR supplementation did not affect VO2max and concentric peak torque, but improved isometric peak torque by 8% (P = 0.048) and the fatigue index by 15% (P = 0.012) in the old. In contrast, NR supplementation did not exert any redox or physiological effect in the young. CONCLUSIONS: NR supplementation increased NAD(P)H levels, decreased oxidative stress, and improved physical performance only in old subjects, substantiating that redox supplementation may be beneficial only in individuals with antioxidant deficiencies.

Correction to: DNA Damage Following Acute Aerobic Exercise: A Systematic Review and Meta-analysis.

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Effect of body composition on redox homeostasis at rest and in response to exercise: The case of underfat women.

Underfat individuals have been neglected as a malnourished population in terms of redox homeostasis. The aim of the present study was to evaluate the effect of body composition on redox homeostasis at rest and in response to exercise. Underfat, lean and overfat women, classified according to their BMI and body fat percentage, participated in the study and were subjected to an acute session of eccentric exercise. With regard to muscle function and damage, a significant group × time interaction was found for range of motion (P < .01), isometric peak torque at 90° (P < .01), delayed onset muscle soreness (P < .01) and creatine kinase (P < .05), with the lean group generally exhibiting faster recovery compared to the underfat and overfat groups. With regard to redox homeostasis, a significant group × time interaction was found for F2-isoprostanes, protein carbonyls and glutathione (P < .01 for all biomarkers), with the underfat and overfat groups exhibiting increased resting oxidative stress levels and lower exercise-induced reactive species production . In conclusively, our data underline the importance of normal body composition for redox homeostasis, since underfat and overfat women demonstrate a similar pattern of redox disturbances both at rest and in response to exercise.

Antioxidants in Personalized Nutrition and Exercise.

The present review highlights the idea that antioxidant supplementation can be optimized when tailored to the precise antioxidant status of each individual. A novel methodologic approach involving personalized nutrition, the mechanisms by which antioxidant status regulates human metabolism and performance, and similarities between antioxidants and other nutritional supplements are described. The usefulness of higher-level phenotypes for data-driven personalized treatments is also explained. We conclude that personally tailored antioxidant interventions based on specific antioxidant inadequacies or deficiencies could result in improved exercise performance accompanied by consistent alterations in redox profile.

A Novel Swimming Performance Test in Rats.

Swimming is an advantageous exercise modality since it induces limited muscle damage. Performance is a crucial endpoint measurement of physiological relevance in exercise physiology and clinical settings alike. To our knowledge, the literature lacks a comprehensive and widely accepted swimming performance protocol without suffering from high variability in time to exhaustion. Thus, the present study presents an easily carried out, two-phased swimming performance incremental test exhibiting low variability in the time to exhaustion among rats. All nine rats managed to complete the first 12 min-part of the test (phase 1) with gradually increased loads attached at the base of their tails equal to 2%, 3.5% and 5% (for 4 min each). All rats reached exhaustion at the 10% final load (phase 2). The mean swimming time until exhaustion, as a measure for defining exercise performance, was 865 ± 59 s. In conclusion, we have presented in detail a novel protocol for practically and satisfactorily measuring swimming performance in rats characterized by low variability in the time to exhaustion. This protocol, with the appropriate modifications, can be applied to a wide spectrum of experimental treatments.