Scand J Med Sci Sports 2014: 24 (Suppl. 1): 43–56 doi: 10.1111/sms.12259

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

A preliminary study: Effects of football training on glucose control, body composition, and performance in men with type 2 diabetes T. R. Andersen1, J. F. Schmidt1,2, M. Thomassen1, T. Hornstrup1, U. Frandsen3, M. B. Randers1, P. R. Hansen2, P. Krustrup1,4, J. Bangsbo1 1

Copenhagen Centre for Team Sport and Health, Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark, 2Department of Cardiology, Gentofte Hospital, Denmark, 3Institute for Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark, 4Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK Corresponding author: Jens Bangsbo, Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, DK 2100 Copenhagen, Denmark. Tel: +0045 35 32 16 23, Fax: +0045 35 32 16 00, E-mail: [email protected] Accepted for publication 29 April 2014

The effects of regular football training on glycemic control, body composition, and peak oxygen uptake (VO2peak) were investigated in men with type 2 diabetes mellitus (T2DM). Twenty-one middle-aged men (49.8 ± 1.7 years ± SEM) with T2DM were divided into a football training group (FG; n = 12) and an inactive control group (CG; n = 9) during a 24-week intervention period (IP). During a 1-h football training session, the distance covered was 4.7 ± 0.2 km, mean heart rate (HR) was 83 ± 2% of HRmax, and blood lactate levels increased (P < 0.001) from 2.1 ± 0.3 to 8.2 ± 1.3 mmol/L. In FG, VO2peak was 11% higher (P < 0.01), and total fat

mass and android fat mass were 1.7 kg and 12.8% lower (P < 0.001), respectively, after IP. After IP, the reduction in plasma glucose was greater (P = 0.02) in FG than the increase in CG, and in FG, GLUT-4 tended to be higher (P = 0.072) after IP. For glycosylated hemoglobin (HbA1), an overall time effect (P < 0.01) was detected after 24 weeks. After IP, the number of capillaries around type I fibers was 7% higher (P < 0.05) in FG and 5% lower (P < 0.05) in CG. Thus, in men with T2DM, regular football training improves VO2peak, reduces fat mass, and may positively influence glycemic control.

The prevalence of type 2 diabetes mellitus (T2DM) is reaching pandemic proportions, with the number of T2DM patients estimated to rise to 366 million by year 2030 (Wild et al., 2007). T2DM is characterized by hyperglycemia, fasting hyperinsulinemia, and insulin resistance in peripheral tissues, and is often related to obesity (Colosia et al., 2013). Glycosylated hemoglobin (HbA1c) is a marker of blood glucose over the preceding 2–3 months and elevated levels of HbA1c are linked to increased risk of cardiovascular disease (CVD) in T2DM (Stratton et al., 2006). Moreover, a 1% absolute lowering in HbA1c is associated with a 15–20% reduction in major adverse cardiovascular end points (Stratton et al., 2000; Selvin et al., 2004). Generally, the chronically elevated blood glucose concentration, low fitness levels, and low levels of daily physical activity in T2DM patients are independently associated with increased all-cause mortality (Wei et al., 2000; Laaksonen et al., 2007). Physical inactivity largely impacts muscle morphology resulting in lowering of fiber size and muscle capillarization, and results in changes in fiber type distribution and mitochondrial content (Hamburg et al., 2007). Conversely, exercise training has repeatedly been

shown to improve muscle function including enhanced insulin responsiveness and GLUT-4 content (Hardin et al., 1995; Holten et al., 2004) as well as improved oxidative capacity and mitochondrial content in skeletal muscle of T2DM subjects (Toledo & Goodpaster, 2013). Thus, alongside glucose-lowering medication and diet, physical activity is a cornerstone of the treatment of T2DM aimed at overall health benefits, improved glycemic control, reductions in HbA1C, and reduced all-cause mortality (Albright et al., 2000; Sigal et al., 2013). At least 150 min of moderate to vigorous aerobic exercise per week or 75 min of vigorous exercise is currently recommended for patients with T2DM (American Diabetes Association, 2013) and may lead to favorable changes in body composition and maximum oxygen uptake (VO2max; Hansen et al., 2010), as well as positive changes in blood glucose control, blood pressure control, and reductions in CVD (Boule et al., 2001; Colberg et al., 2010). When any type of exercise was evaluated in a meta-analysis, structured exercise training was associated with a reduction of 0.6% in HbA1c levels (Thomas et al., 2006). More specifically, the reductions in HbA1c levels after training periods lasting less than 6 months

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Andersen et al. were more pronounced with aerobic training (0.46%) than with resistance training (0.32%; Yang et al., 2013), which would appear to be a considerable additional effect to the 1.12% reduction reported with antidiabetic treatment with metformin alone (Hirst et al., 2012). Despite the known therapeutic effects of exercise training, patients with T2DM are reluctant to take up regular exercise (Donahue et al., 2006). Primary perceived barriers include lack of time, poor adherence, and low self-efficacy in the ability to implement exercise. Football is well-known to most people all over the world, and it may motivate for both initiation and maintenance of lifelong physical activity through elevated social capital emerging from the sport-dependent psychosocial interactions (Ottesen et al., 2010). In recent years, the health benefits of recreational football training in various populations have been investigated. Interestingly, the physiological load during recreational football training (Randers et al., 2010b) is similar to what observed in high-level male elite players (Impellizzeri et al., 2006), and 1 h of football training 2–3 times per week for 12 weeks has been shown to improve VO2max, cardiovascular risk profile, and muscle mass and reduce body fat percentage in a group of untrained males (Krustrup et al., 2009). Therefore, football may be an attractive high-intensity training alternative to interventions traditionally applied for nonpharmacological prevention and treatment of T2DM. Recently, we observed marked effects of football training on cardiac function and blood pressure in T2DM patients (Schmidt et al., 2013), but whether football training has a significant effect on glycemic control, body composition (adiposity) and muscular adaptations warrants investigation. Thus, the aim of the present study was to evaluate the effects of 24 weeks of recreational football training on glycemic control, muscular adaptations, body composition, and maximal oxygen uptake in middle-aged male subjects with T2DM. Materials and methods Subjects Twenty-one male subjects with type 2 diabetes mellitus (T2DM) were recruited, and assigned to either a football training group (FG; n = 12) or an inactive control group (CG; n = 9). Medical screening was performed before the start of the intervention period (IP). Inclusion criteria were no changes in antidiabetic medication for a 3-month time period, no history or symptoms of cardiovascular disease or cancer, absence of diabetic complications [nephropathy (plasma creatinine > 90 µmol/L), retinopathy, and neuropathy], type 1 DM, treatment with beta-blockers (due to their heart rate-lowering properties), or musculo-skeletal complaints that were considered to interfere with football training. The study aimed to conduct a randomized clinical controlled trial. However, external factors such as traveling distances to the training facilities, and subjects requesting allocation to either group made randomization unattainable. Despite this, no group differences (FG vs CG) were observed in the pre-intervention values for age (50.6 ± 7.1 vs 48.7 ± 9.2 years), duration of T2DM (7.1 ± 2.2 vs 7.5 ± 3.6 years), homeostatic model assessment of diabetes insulin

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resistance (HOMA-IR; 4.9 ± 3.0 vs 5.3 ± 2.0), body mass index (BMI; 30.4 ± 3.4 vs 30.4 ± 6.7 kg/m2) and VO2peak (30.5 ± 2.9 vs 27.5 ± 8.2 mL/min/kg). Details of subject recruitment and randomization have been described elsewhere (Schmidt et al., 2013). All subjects were informed of potential risks and discomforts associated with the experimental procedures before giving their written informed consent to participate. The study conformed to the code of ethics of the World Medical Association (Declaration of Helsinki) and was approved by the local ethical committee of Copenhagen (H-2-2011-088). The study was reported at ClinicalTrials.gov.: NCT01636349.

Experimental design The study represented an independent part of a comprehensive interventional protocol investigating cardiovascular and muscular adaptations as well as changes in physical performance and health status in the study participants. Data on cardiovascular adaptations have been published in a companion paper (Schmidt et al., 2013). The subjects in FG performed 60 min of supervised football training twice a week for 24 weeks. Subjects in CG were instructed to continue their sedentary lifestyle. All subjects were examined before IP (0 weeks) and after 12 and 24 weeks. All subjects completed testing after 12 weeks, whereas two subjects in FG and one subject in CG left the study before completing the testing sessions at week 24. Individual reasons for leaving the study were not obtained. The testing protocol included muscle biopsies, blood samples, dual energy X-ray absorptiometry (DXA) scanning, and cycling testing (see below). In FG, measurements were also performed during a selected session to establish the metabolic response to training.

Medication and daily physical activity level During IP, the subjects maintained their routine glucose-lowering treatment and habitual daily physical activity levels, and were instructed not to change lifestyle. Also, the participants were instructed to report any changes in medication prescriptions during the study period. Data on habitual daily physical activity level and medication have recently been reported (Schmidt et al., 2013) with no differences between FG and CG.

Training intervention In FG, 1 h of supervised football training was performed twice a week for 24 weeks. The training sessions consisted of small-sided (four-a-side, five-a-side, six-a-side) games played on a 20-m-wide and 40-m-long indoor court surrounded by walls. The subjects played five times 10-min games interspersed with 2 min of passive rest. The average total number of training sessions during IP was 37.6 (1.5 sessions per week). The average weekly training attendance was the same during the initial 12 weeks and the last 12 weeks of the intervention period (1.56 ± 0.09 (± SEM) vs 1.47 ± 0.10 sessions per week). The average effective playing time was the same (P = 0.072) during the first 12 weeks and the last 12 weeks of IP (44 ± 2 vs 48 ± 5 min/session, respectively).

Measurements and test procedures Maximal cycling testing Before as well as after 12 and 24 weeks of IP, pulmonary gas exchange (OxyconPro; VIASYS Healthcare, Hoechberg, Germany) and heart rate (HR; Polar Team System, Polar Electro Oy, Kempele, Finland) were measured during a standardized cycling protocol. Subjects started exercising at a work pace and load of 80 rpm and 40 W, respectively. Thereafter, the workload was

Football training and T2DM increased by 20 W every 2 min until volitional fatigue. A leveling off in oxygen uptake with an increase in work rate at the end of the test was used to objectively confirm achievement of VO2peak. VO2peak was determined as the highest value achieved during a 30 s period. Timeto-exhaustion was noted, and maximal heart rate (HRmax) was determined as the highest value measured during the test. Subjects were familiarized with all procedures prior to the testing sessions at the beginning of IP. No strenuous physical activities were performed 2 days before a testing session, and intake of caffeine and alcohol on the day of the experiment was avoided.

period, and 10-min into recovery from the previous playing period. The subjects were weighed wearing dry shorts before, and immediately after the training session using a digital scale (OHAUS 1-10, Pine Brook, New Jersey, USA) to determine sweat loss. The subjects were allowed to drink water ad libitum during the test and the water intake was recorded. Perceived exertion was recorded using visual analog scale (VAS) questionnaires. VAS questionnaires were collected 15–30 min after the end of the training session to ensure that the perceived effort referred to the whole session rather than the most recent exercise (Impellizzeri et al., 2004).

Muscle biopsy and blood sample collection

Time-motion analysis procedure

All invasive procedures were performed 48–72 h after a training session, between 7:00 and 10:00 h, and under standardized conditions after an overnight fast. A blood sample was collected from an antecubital vein, and a biopsy was collected at rest after 0, 12, and 24 weeks from m. vastus lateralis under sterile conditions and local anesthesia (1% Lidocaine, Amgros 742122, Copenhagen, Denmark) using the Bergstrom technique (Bergstrom, 1962). A part of the muscle sample (40 mg wet weight) was immediately frozen in liquid N2 and stored at −80 °C. The remainder of the muscle tissue was mounted in an embedding medium (OCT Tissue-Tek, Sakura Finetek, Zoeterwoude, the Netherlands) and frozen in precooled isopentane and subsequently stored at −80 °C until further analysis (see below).

The video recordings were replayed on a monitor for computerized coding of the activity pattern (Bangsbo, 1994). Markers on the floor were used to calculate the speed for the locomotor categories categorized as: standing (0 km/h), walking (4 km/h), jogging (8 km/h), moderate-speed running (14 km/h), high-speed running (18 km/h), and backwards running (10 km/h). Also, the numbers of tackles, in-fights, jumps, passes of the ball, and goal-scoring attempts during the training session were recorded.

Body composition After 0, 12, and 24 weeks, whole body and regional fat mass, lean mass, and bone mass were determined by whole-body DXA scanning (Prodigy Advance, Lunar Corporation, Madison, Wisconsin, USA). Scanning was performed between 7:00 and 10:00 h under standardized conditions after an overnight fast. Additionally, body height and body weight were measured with subjects wearing light clothes and BMI (kg/m2) was calculated. Measurements during training In FG, HR was recorded at 1-s intervals with a HR monitor during each training session (Polar Oy, Kempele, Finland). Resting periods were filtered out and only effective playing time during training was analyzed. On one occasion, eight subjects were individually videoed with digital cameras (GR-D23E, JVC, Tokyo, Japan) during a training session for subsequent timemotion analysis. In addition, to establish the metabolic response to recreational indoor football training, the subjects had a catheter placed in an antecubital vein before the training session for concurrent collection of blood samples. Blood samples were collected immediately before the start of the training, after each 10-min playing

Blood analyses Whole blood samples were analyzed for basal levels of glycosylated hemoglobin (HbA1c) using liquid chromatography (TOSOH G7). Basal plasma levels of glucose, insulin and C-peptide were analyzed using automated procedures (Modular P-module, Modular E-module, and Modular E-module, respectively; Cobas Fara, Roche, Neuilly sur Seine, France) and standard reagents (Glu11876899, Insulin-12017547122, and C-peptide – 03184897190; Cobas Fara, Roche, Neuilly sur Seine, France) at the clinical laboratory at Rigshospitalet, Copenhagen, Denmark, according to the manufacturers descriptions. HOMA-IR and HOMA C-peptide was calculated using the Oxford calculator (www.dtu.ox.ac.uk/ homacalculator/). A part of the blood sample was rapidly centrifuged for 90 s. From this, plasma was collected and stored at −20 °C and subsequently analyzed for plasma fibroblast growth factor (FGF) 21 using an alphaLISA kit (PerkinElmer, Waltham, Massachusetts, USA; intraand inter-assay variation CV being 7.9% and 12.8%, respectively). Soluble intercellular adhesion molecule-1 (sICAM-1; Emax precision microplate reader, Molecular Devices, Sunnyvale, California, USA), and endothelin-1 (ET-1; Orion L Microplate Luminometer, 300–600 nm, Titertek-Berthold, Huntsville, Alabama, USA) were measured using an immunoassay (ELISA, R&D Systems, Inc., Minneapolis, Minnesota, USA). Whole blood samples collected during training were immediately stored on ice and subsequently analyzed for blood lactate and blood glucose using an ABL 800 Flex

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Andersen et al. (Radiometer, Copenhagen, Denmark). Also, part of this blood sample was rapidly centrifuged for 90 s. Plasma was collected and stored at −20 °C, and subsequently analyzed for plasma free fatty acids (FFA) using a fluorometric enzymatic kit (WAKO Chemicals GmbH, Neuss, Germany). Muscle analyses Maximal enzyme activity The frozen samples were weighed before and after freeze-drying to determine water content. The samples were then dissected free of all visual connective tissue and blood by light microscopy (Stemi 2000-C, Zeiss, Oberkochen, Germany) at a room temperature of −18 °C and a relative humidity below 30%. The muscle tissue of dry weight samples was analyzed for citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activity using flourometric methods with NAD-NADHcoupled reactions (Lowry & Passonneau, 1972). Immunofluorescence microscopy Muscle fiber cross-sectional area, fiber type distribution, and capillarization were analyzed using a staining protocol procedure previously described (Nielsen et al., 2012). The embedded muscle samples were cut using a cryostat, and transverse sections 8 µm in thickness were placed onto glass slides. To verify the cross-sectional orientation of the individual muscle fiber, multiple samples were cut and examined under light microscopy until at cross-section of desirable size, orientation, and uniform polygonal appearance was visible. Only areas without artifacts or tendency to longitudinal cuts were

used analyses. Staining targets were visualized pair wise. Firstly, capillaries and myofiber type IIA were visualized using biotinylated Ulex europaeus agglutinin I lectin (Ulex; VECTB-1065, VWR, Bie & Berntsen, Herlev, Denmark, 1:100) and an monoclonal antibody (SC-71, Hybridoma Bank, Iowa City, Iowa, USA, 1:500), respectively. Secondly, myofiber borders were visualized using an antibody against laminin (Dako Z0097, Glostrup, Denmark, 1:1000), together with myosin heavy chain (MHC)-s (Sigma, M8421, 1:1000) added for distinction of myofiber type slow/type I. Specific secondary antibodies (order listed: Streptavidin/FITC, Dako F0422, Glostrup, Denmark), Alexa-555 donkey anti-mouse (Invitrogen, A31570, Life Technologies Denmark, Naerum, Denmark, 1:1000), Alexa-350 goat anti-rabbit (Invitrogen, P10994, 1:1000) and Alexa-488 donkey anti-mouse (Invitrogen, A21202, 1:1000) were applied to each primary antibody. Specificity of the staining was assessed by single staining, and by staining without the primary antibody. Three individual muscle fiber types were identified as types I (green), IIA (red), and IIX (unstained/black; Bloemberg & Quadrilatero, 2012; Fig. 1). Visualization was performed on a computer screen using a light microscope (Carl Zeiss Axio Imager M1, Zeiss, Oberkochen, Germany), and all morphometric analyses were performed using a digital analysis program (Carl Zeiss, AxioVision 4.6). Two or more separate sections of a cross-section were used for analyses, and the cross-sectional area was assessed by manually drawing the perimeter around each selected section. The number of muscle fibers and capillaries within each section was counted, and capillary supply was subsequently expressed as capillaries per fiber (C : F-ratio), capillary density (capillaries/mm) and capillaries around

Fig. 1. Representative immunohistochemical staining containing laminin (blue), MHC-s/Ulex (green), and SC-71 (red). Scale bar = 100 µm.

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Football training and T2DM a fiber (CAF). A mean of 127 myofibers (range: 76–235) were analyzed per biopsy, and mean fiber area was assessed by manual drawing of the perimeter of each muscle fiber. All analyses were carried out manually by the same blinded investigator (T. R. A.). Protein expression in muscle homogenate lysates Changes in protein expression were determined by a standard western blotting procedure as previously described in detail by our laboratory (Thomassen et al., 2010). In short, total protein concentration in each sample was determined by a BSA standard kit (Pierce Biotechnology Inc., Rockford, IL, US) and samples were mixed with 6 × Laemmli buffer (7 mL 0.5M Tris-base, 3 mL glycerol, 0.93 g DTT, 1 g SDS, and 1.2 mg bromophenol blue) and ddH20, and from each muscle biopsy, two samples were made, and samples from the same subject were loaded on the same gel. The intensity of each band were first normalized to the mean intensity of two human standard lysates loaded on the same gel and then normalized to the PRE group mean value. As previously described (Nordsborg et al., 2008), outliers were identified and excluded. The exclusion criteria were set to ratio changes from pre- to 12 weeks (mid) and from – to 24 weeks (post) +/−2 SD away from the group mean. As ratios are, by definition, not normal distributed and were log transformed before the statistical analyses and then back transformed to geometric means with the 95% confidence interval. Changes in percent were obtained as the differences in geometric means. To determine specific protein expression the following antibodies were used with the localization of the quantified signal noted: total actin: 42 kDa, rabbit (A2066, Sigma-Aldrich A2066, Brøndby, Denmark); Akt1: 60 kDa, mouse (#2967); Akt2: 60 kDa, rabbit (#3063) and glycogen synthase (GS): 84 kDa, rabbit (#3893) (Cell Signalling Technology, Danvers, MA, US); CS: 48 kDa, rabbit (ab96600) and HAD: 83 kDa, rabbit (ab54477) (Abcam, Cambridge, UK); cytochrome C oxidase subunit IV (COX4): 17 kDa, mouse (sc58348) and platelet/endothelial cell adhesion molecule-1 (PECAM-1 or CD31): 130 kDa, rabbit (sc-1506R) (Santa Cruz Biotechnology, Dallas, TX, US); Glucose Transporter 4 (GLUT4): 46 kDa, rabbit (PA1-1065, Thermo Scientific Hvidovre, Denmark); mitochondrial complex I-V: mitochondrial complex I subunit NDUFB8 (CI): 20 kDa (ab110242), mitochondrial complex II, succinate dehydrogenase complex subunit B (CII): 30 kDa (ab14714), mitochondrial complex III subunit core 2 (CIII): 45 kDa (ab14745), mitochondrial complex IV – cytochrome C oxidase subunit II (CIV): 25 kDa (ab110258), mitochondrial complex V ATP synthase subunit alpha (CV): 55 kDa (ab14748) all five mouse and included in the MitoProfile® Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam, Cambridge, UK). The secondary antibodies used were

HRP conjugated goat anti-mouse (P-0447 DAKO, Glostrup, Denmark) and goat anti-rabbit (4010-05 SouthernBiotech, Birmingham, AL, US). Statistics Group differences before IP were analyzed using a twotailed unpaired t-test. HR and blood data obtained during a training session were analyzed using a one-way repeated measures analysis of variance (RM ANOVA). Pearson product placement was used to test associations between selected variables. Between-group and withingroup changes after 12 and 24 weeks were analyzed using a two-way RM ANOVA. In the data analysis of FGF-21, an analysis of covariance (ANCOVA) model was additionally applied to correct for age, BMI, total body fat mass, fasting plasma glucose, fasting plasma insulin, blood lipids, and cholesterol, VO2max, and baseline levels in FGF-21. Changes in protein expression were determined using one-way RM ANOVA separately for FG and CG, based on data being expressed in arbitrary units making comparisons between groups difficult to interpret. When a significant time-by-group effect could be detected a Student-Newman-Keuls post-hoc analysis was applied to determine differences between different time points. There were three study dropouts from 13–24 weeks, and for missing values the last observation carried forward method was used. All analyses were controlled with two-way RM ANOVA without imputations and no relevant statistical differences were found. P < 0.05 was chosen as the level of significance. Statistical analyses were performed using Sigma plot, version 11.0 (Systat Software Inc., San Jose, CA, US) and R (www.r-project.org). Results Physiological response to a training session Mean HR during a training session was 149 ± 4 bpm (mean ± SEM), corresponding to 83 ± 2% of HRmax (HRmax; 179 ± 2 bpm). Time spent in heart rate zones corresponding to < 70% HRmax, 70–80% HRmax, 80–90% HRmax, and 90–100% HRmax during play was 5 ± 2, 30 ± 9, 54 ± 10, and 11 ± 9% of total playing time, respectively (Fig. 2(a)). Individual peak heart rate was 166 ± 3 bpm corresponding to 92 ± 1% HRmax. The subjects performed 707 ± 40 changes of activity corresponding to a change of activity approximately every 4 s. The total distance covered was 4692 ± 197 m, and walking, jogging, moderate-intensity running, highintensity running, and backwards running accounted for 2017 ± 72, 1062 ± 76, 1332 ± 168, 209 ± 36, and 72 ± 12 m, respectively (Fig. 2(b)). In addition, subjects performed 1.0 ± 0.4 jumps, 53 ± 6 passes, 15 ± 2 attempts on goal, and 8 ± 1 tackles, and were engaged in 1.4 ± 0.4 in-fights during a training session. Blood lactate values increased (P < 0.001) from 2.1 ± 0.3 to 8.2 ± 1.3 mmol/L after 10 min of training,

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Andersen et al. (a)

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Fig. 2. Heart rate distribution (a) and distance covered in various locomotor modes (b): walking (W), jogging (J), moderate-speed running (MS), high-speed running (HS), and backwards running (Br), as well as blood lactate (c), and plasma FFA levels (d) before, during and after a training session in male type 2 diabetic subjects (n = 8). Mean and individual values are presented.

and remained elevated throughout the training session (Fig. 2(c)). Plasma FFA did not change during the training, but was elevated 10 min after the session (Fig. 2(d)). Individual mean blood lactate levels throughout the training session correlated (r = 0.82, P = 0.01) with the proportion of total playing time spent at moderateintensity running, high-intensity running, and sprinting. Rating of perceived exertion (VAS score) was 73 ± 14% of maximal value. Net weight loss during training was 0.44 ± 0.10 kg.

Peak oxygen uptake In FG, VO2peak after 12 and 24 weeks of training was 10% and 11% higher (3.22 ± 0.13 and 3.26 ± 0.16 L/min, P < 0.001), respectively, compared with before IP (2.92 ± 0.18 L/min), while in CG it did not change (Fig. 3). At exhaustion, respiratory exchange ratio (RER) was the same (P > 0.05) after 0, 12, and 24 weeks being 1.11 ±

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0.01, 1.12 ± 0.01, 1.10 ± 0.02, respectively, in FG, and 1.12 ± 0.03, 1.11 ± 0.02, 1.13 ± 0.03, respectively, in CG.

Body composition In FG, total fat mass was 3.7% (P < 0.01) and 5.7% (P < 0.001) lower after 12 and 24 weeks, respectively, compared with before IP, corresponding to a decrease in total fat mass of 1.1 and 1.7 kg (Fig. 4(a)), and 3.3% (P < 0.01) and 4.9% (P < 0.001) lower total body fat percentage after 12 and 24 weeks, respectively, compared with before IP (Table 1). In FG, android fat mass was 7.6% (P < 0.01) and 12.8% (P < 0.001) lower after 12 and 24 weeks, respectively, compared with before IP, corresponding to a 3.4% (P < 0.01) and 5.2% (P < 0.001) lower android fat percentage (Fig. 4(b)). In CG, total fat mass, total body fat percentage, android fat mass, and android fat percentage did not change (P > 0.05). In FG, leg lean mass, and leg bone mass did

Football training and T2DM

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Fig. 3. Peak oxygen consumption in male type 2 diabetic subjects after 0, 12, and 24 weeks of football training (FG; open bars) and continuation of an inactive lifestyle (CG; hatched bars). Means ± SEM are presented. *Significant (P < 0.05) different from 0 weeks.

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Fig. 4. Total body fat mass (a) and android fat percentage (b) in male type 2 diabetic subjects after 0, 12 and 24 weeks of football training (FG; open bars) and continuation of an inactive lifestyle (CG; hatched bars). Means ± SEM are presented. **Significantly (P < 0.01) different from 0 weeks. ***Significantly (P < 0.001) different from 0 weeks. Mean and individual values are presented.

not change, whereas in CG, leg lean mass and leg bone mass were 3.5% and 1.6% lower (P < 0.05), respectively, after 24 weeks compared with before IP.

ET-1, FGF-21, and calculated HOMA C-peptide, and HOMA-IR did not change during IP (Table 2). Muscle variables

Blood variables A significant time-by-group interaction was observed for resting blood glucose after 24 weeks compared with before IP. In FG, resting blood glucose was nonsignificantly (P = 0.071) reduced by 9.4% in FG, while in CG it was nonsignificantly (P = 0.23) 9.8% higher (Table 2). A significant (P < 0.01) time effect was detected for HbA1c after 24 weeks compared with before IP (Table 2). Plasma insulin, C-peptide, s-ICAM,

Before IP, the number of capillaries distributed around type I muscle fibers was lower (P < 0.01) in FG than in CG (4.21 ± 0.20 vs 4.93 ± 0.21 CAF). A significant (P < 0.05) time-by-group interaction was detected for capillaries around type I fibers, increasing by 7% in FG, and decreasing by 5% in CG (P < 0.05; Table 3). Fiber area of muscle fiber type I, type IIA, and type IIX, as well as mean fiber area, and fiber type distribution remained the same during IP (Table 3).

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50 3.26 ± 0.16*** 34.1 ± 3.3***‡ 94.3 ± 4.2 30.1 ± 1.0 27.8 ± 2.0*** 29.2 ± 1.2*** 3.4 ± 0.3*** 41.2 ± 1.5*** 3.8 ± 0.2 27.9 ± 1.2 1.5 ± 0.1 63.2 ± 2.6 21.8 ± 1.0 1293 ± 43 1.497 ± 0.031

2.73 ± 0.20 27.5 ± 2.5 100.4 ± 6.1 30.4 ± 2.2 31.0 ± 5.3 29.4 ± 3.7 3.5 ± 0.6 38.8 ± 4.5 4.7 ± 0.8 30.4 ± 3.6 1.3 ± 0.1 66.0 ± 2.4 22.4 ± 1.1 1381 ± 78 1.472 ± 0.056

3.22 ± 0.13*** 34.0 ± 2.5***‡ 94.6 ± 4.3 30.1 ± 1.0 28.4 ± 2.1** 29.7 ± 1.2** 3.6 ± 0.3*** 42.0 ± 1.4** 4.0 ± 0.3 28.6 ± 1.3 1.5 ± 0.0 63.0 ± 2.6 21.7 ± 1.0 1282 ± 41 1.505 ± 0.035

2.92 ± 0.18 30.5 ± 2.9 95.4 ± 4.3 30.4 ± 1.0 29.5 ± 2.0 30.7 ± 1.1 3.9 ± 0.3 43.5 ± 1.3 4.1 ± 0.3 28.8 ± 1.1 1.5 ± 0.0 62.5 ± 2.5 21.3 ± 1.1 1279 ± 42 1.497 ± 0.031

Means ± SEM are presented. *Significantly (P < 0.05) different from 0 weeks. **Significantly (P < 0.01) different from 0 weeks. ***Significantly (P < 0.001) different from 0 weeks. † Significantly (P < 0.05) different from 12 weeks. ‡ Significantly (P < 0.01) different from CG. § As previously reported by Schmidt et al., 2013 (with permission). ANOVA, analysis of variance; BMD, bone mineral density; CG, control group; FG, football training group.

VO2peak (L/min) VO2peak (mL/min/kg)§ Total body mass (kg) Body mass index (kg/m2) Total fat mass (kg) Body fat percentage (%) Android fat mass (kg) Android fat percentage (%) Gynoid fat mass (kg) Gynoid fat percentage (%) A/G-ratio Total lean mass (kg) Leg lean mass (kg) Leg bone mass (g) Leg BMD (g/cm2)

0 weeks

12 weeks

0 weeks

24 weeks

CG

FG

2.83 ± 0.27 28.1 ± 2.8 101.0 ± 6.2 30.6 ± 2.3 30.9 ± 5.2 29.2 ± 3.7 3.5 ± 0.6 38.6 ± 4.4 4.7 ± 0.8 30.4 ± 3.5 1.3 ± 0.1 66.7 ± 2.6 22.7 ± 1.2 1377 ± 81 1.486 ± 0.059

12 weeks 2.79 ± 0.24 27.7 ± 2.5 101.0 ± 6.6 30.7 ± 2.4 30.9 ± 5.2 29.2 ± 3.5 3.5 ± 0.6 38.8 ± 4.4 4.7 ± 0.9 30.1 ± 3.5 1.3 ± 0.1 66.8 ± 2.7 21.9 ± 1.1*† 1354 ± 74*† 1.452 ± 0.057

24 weeks

< 0.001 < 0.001 0.864 0.926 0.005 0.004 < 0.001 < 0.01 0.054 0.233 0.444 0.119 0.150 0.540 0.295

Time

ANOVA

0.225 0.049 0.419 0.866 0.652 0.862 0.802 0.408 0.375 0.578 0.119 0.342 0.624 0.313 0.627

Group

0.027 < 0.01 0.054 0.050 0.013 0.046 < 0.001 < 0.01 0.251 0.694 0.561 0.927 0.025 0.006 0.577

Group × time

Table 1. Peak oxygen uptake and anthropometric characteristics in male type 2 diabetic subjects before (0 weeks) and after 12 and 24 weeks of football training (FG) or continuation of an inactive lifestyle (CG)

Andersen et al.

Football training and T2DM Table 2. Plasma s-ICAM-1, plasma endothelin-1, plasma fibroblast growth factor-21, insulin, resting blood glucose, and blood C-peptide in male type 2 diabetic subjects before (0 weeks) and after 12 and 24 weeks of football training (FG) or continuation of an inactive lifestyle (CG)

FG

sICAM-1 (ng/mL) ET-1 (pg/mL) FGF-21 (pg/mL) Insulin (pmol/L) Glucose (mmol/L) HbA1c (mmol/L) C-peptide (mmol/L) HOMA-IR HOMA C-peptide

CG

ANOVA

0 weeks

12 weeks

24 weeks

0 weeks

12 weeks

24 weeks

Time

Group

Group × time

202.9 ± 19.9 2.2 ± 0.7 2815 ± 344 54.0 ± 9.0 8.8 ± 0.7 7.4 ± 0.3 0.90 ± 0.09 3.3 ± 0.6 2.5 ± 0.2

190.1 ± 14.2 2.3 ± 0.5 2741 ± 371 52.7 ± 8.9 8.0 ± 0.6 6.9 ± 0.3 0.90 ± 0.08 2.6 ± 0.4 2.3 ± 0.2

190.6 ± 19.0 1.9 ± 0.2 2809 ± 402 47.3 ± 8.1 7.8 ± 0.6 7.0 ± 0.3 0.88 ± 0.08 2.4 ± 0.4 2.2 ± 0.2

176.9 ± 18.4 3.0 ± 0.9 5647 ± 1988 63.6 ± 8.9 7.6 ± 0.7 7.5 ± 0.4 0.92 ± 0.14 2.9 ± 0.5 2.7 ± 0.3

176.6 ± 17.3 2.5 ± 0.4 5989 ± 2258 67.3 ± 10.8 8.4 ± 0.9 7.4 ± 0.4 0.94 ± 0.16 3.7 ± 0.6 2.6 ± 0.3

176.2 ± 18.0 2.1 ± 0.5 5800 ± 2048 62.3 ± 6.2 8.5 ± 1.2 7.5 ± 0.4 0.99 ± 0.15 3.7 ± 0.6 2.9 ± 0.3

0.652 0.598 0.955 0.990 0.963 0.007 0.945 0.998 0.675

0.467 0.361 0.001 1.000 0.970 0.732 0.878 1.000 0.640

0.693 0.846 0.851 0.982 0.020 0.210 0.084 0.971 0.082

Means ± SEM are presented. ANOVA, analysis of variance; CG, control group; ET-1, endothelin-1; FG, football training group; FGF-21, fibroblast growth factor-21; HOMA-IR, homeostatic model assessment of diabetes insulin resistance; sICAM-1, soluble intercellular adhesion molecule-1.

No change in CS and HAD activity was observed during IP in either FG or CG (Table 3), whereas in FG, CS expression was lower (P < 0.05) after 12 (7%) and 24 (10%) weeks compared with before IP with no change observed for CG (Fig. 5(a)). In FG, mitochondrial complex I expression was 9% lower (P < 0.05) after IP (0.98 (0.87–1.10) vs 0.89 (0.79–1.00) geometric mean (95% CI) for 0 weeks and 24 weeks, respectively), with no change observed in CG (Fig. 5(b)). In FG, GLUT-4 expression was nonsignificantly (P = 0.072) elevated after IP [0.93 (0.75–1.16) vs 1.17 (0.95–1.44)], while GLUT-4 expression did not change in CG (Fig. 5(c)). In FG, Akt-2 protein expression was 24% higher (P < 0.05) after 24 weeks [1.20 (1.03–1.40)] compared with 0 weeks [0.96 (0.81–1.14)]. In CG, Akt-2 expression was 35% higher (P < 0.05) after 24 weeks [1.33 (1.10–1.60)] compared with 0 weeks [0.98 (0.86–1.12)] (Fig. 5(d)). In FG, actin protein expression was 14% and 15% higher (P < 0.01) after 12 weeks [1.12 (1.02–1.23)] and 24 weeks [1.12 (0.95– 1.33)], respectively, compared with 0 weeks [0.98 (0.86– 1.11)], whereas no change was observed for actin expression in CG (Fig. 5(e)). For both FG and CG, no changes were observed in content of HAD, glycogen synthase, mitochondrial complexes II, III, IV (subunit 2 and 4), and V, and CD-31 (data not shown). Discussion The major findings of the present study were that regular football training performed as small-sided games in men with T2DM led to increases in VO2peak, lowered total body fat mass, and android fat percentage as well as favorable changes in glycemic control. Furthermore, leg lean mass and leg bone mass were maintained in FG, whereas CG showed a significant reduction during IP. A marked number of muscle and blood variables, such as oxidative enzymes and FGF-21, respectively, were reduced or not changed, although the training signifi-

cantly stimulated both the aerobic and anaerobic energy systems, as evaluated from the high heart rates and accumulation of blood lactate during football training. After 24 weeks, a significant time-by-group interaction was found for fasting glucose levels with the reduction in blood glucose in FG being larger than the increase for CG (Table 2). Also, the number of capillaries surrounding the type I muscle fibers was elevated after 24 weeks in FG (Table 3). Thus, the improved glycemic control observed in FG may be related to an increased extracellular delivery of glucose, which is affected by the blood flow through the muscle and the degree of microvascular perfusion (Clark, 2008). It is, however, not necessarily so that more capillaries will lead to higher blood flow to the muscle. The lowering of blood glucose as a result of the football training may also have been caused by the elevated expression of GLUT-4, even though only a tendency (P = 0.07) was observed (Fig. 5(c)). GLUT-4 is considered to be the most important glucose transporter in skeletal muscle, and is thought to be responsible for both insulin-stimulated and contraction-induced glucose uptake (Dela et al., 1994; Daugaard & Richter, 2001). Football training did not lead to a significant time-by-group interaction for HOMA C-peptide and HbA1c (Table 2), which may be due to insufficient statistical study power and large intra-individual variation in these parameters. As such, calculated odds ratio of having a positive outcome (lowering of HbA1c) after IP was 2.3 (95% CI: 0.34 to 16.2) in FG compared with CG. The observed 0.4% reduction in HbA1c in FG is within the range of the effect of physical training estimated in recent meta-analyses on physical activity of data from T2DM patients (Thomas et al., 2006; Yang et al., 2013). It is likely of clinical relevance in the treatment of T2DM and may contribute to reduced micro- and macrovascular complications (Holman et al., 2008), even if the reduction was not reflected through a statistically significant time-by-group interaction. Thus, football training may represent an attractive alternative to other training

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CS (µmol/g dw/min) HAD (µmol/g dw/min) density (cap/mm2) density (C : F-ratio) Type I (CAF) Type IIA (CAF) Type IIX (CAF) Type I (µm2) Type IIA (µm2) Type IIX(µm2) Mean (µm2) % Type I % Type IIA % Type IIX

32.7 ± 2.0 18.5 ± 0.8 296 ± 14 1.92 ± 0.13 4.21 ± 0.22 3.92 ± 0.23 3.35 ± 0.22 5133 ± 271 5330 ± 343 4651 ± 299 5038 ± 275 43.1 ± 2.8 43.3 ± 2.9 13.6 ± 1.0

30.8 ± 1.4 16.6 ± 0.8 302 ± 8 2.10 ± 0.11 4.41 ± 0.12 4.10 ± 0.13 3.66 ± 0.16 5363 ± 278 6097 ± 298 5649 ± 331 5703 ± 252 41.8 ± 3.9 41.6 ± 3.5 16.5 ± 1.6

12 weeks 30.3 ± 1.0 17.5 ± 0.9 308 ± 10 2.06 ± 0.10 4.52 ± 0.17 4.09 ± 0.17 3.47 ± 0.16 5353 ± 253 5728 ± 353 5436 ± 296 5506 ± 262 43.9 ± 4.1 42.0 ± 3.6 14.1 ± 1.7

24 weeks 28.7 ± 5.0 17.3 ± 1.7 361 ± 19 1.88 ± 0.11 4.93 ± 0.24 4.18 ± 0.16 3.41 ± 0.20 4979 ± 270 4724 ± 263 4151 ± 238 4619 ± 238 42.0 ± 5.0 43.7 ± 5.1 14.3 ± 1.1

0 weeks

CG

Actin protein (AU)

Akt-2 protein (AU)

GLUT-4 protein (AU)

Complex I protein (AU)

0.703 0.096 0.409 0.649 0.049 0.948 0.327 0.564 0.339 0.221 0.350 0.260 0.323 0.708

Group × time

Citrate synthase protein (AU)

0.761 0.864 0.040 0.369 0.093 0.330 0.187 0.232 0.049 0.007 0.023 0.948 0.405 0.782

0.957 0.228 0.857 0.814 0.796 0.660 0.885 0.933 0.136 0.068 0.176 0.610 0.131 0.261

31.4 ± 4.0 17.5 ± 1.4 339 ± 14 1.91 ± 0.32 4.47 ± 0.15 4.26 ± 0.16 3.21 ± 0.14 4770 ± 268 5320 ± 253 4578 ± 271 4856 ± 215 38.9 ± 5.1 34.4 ± 4.9 15.7 ± 2.8

29.1 ± 4.4 20.0 ± 1.9 337 ± 38 1.82 ± 0.14 4.77 ± 0.21 4.27 ± 0.26 3.19 ± 0.19 4888 ± 357 4903 ± 404 4191 ± 320 4661 ± 310 47.0 ± 4.4 37.1 ± 3.5 15.9 ± 2.1

Group

Time

24 weeks

12 weeks

ANOVA

(b)

Means ± SEM are presented. ANOVA, analysis of variance; CAF, capillaries around a fiber; CG, control group; CS, citrate synthase; FG, football training group; HAD, 3-hydroxyacyl-CoA dehydrogenase.

Fiber type distribution

Fiber area

Capillary

Enzyme activity

0 weeks

FG

Table 3. Muscle enzyme activity, capillarization, fiber area, and fiber type distribution in male type 2 diabetic subjects before (0 weeks) and after 12 and 24 weeks of football training (FG) or continuation of an inactive lifestyle (CG)

Andersen et al. (a)

1.4 48 kDa

1.2

1.0

1.4

1.2

∗

1.2

1.0

1.6

1.4

1.6

1.4

∗∗

∗

0.8

1.4 20 kDa

∗

0.8

(c) 46 kDa

(∗ )

1.2

1.0

0.8

(d)

∗ 60 kDa

∗

1.2

1.0

0.8

(e) 42 kDa

∗∗

1.0

0.8

0 weeks 12 weeks 24 weeks 0 weeks 12 weeks 24 weeks

Fig. 5. Muscle protein expression of citrate synthase (a), complex I (b – lower band), Glut-4 (c – upper band), Akt-2 (d – two bands analyzed), and actin (e) in male type 2 diabetic subjects after 0, 12, and 24 weeks of football training (FG; gray bars) and continuation of an inactive lifestyle (CG; open bars). Geometric means and 95% confidence interval are presented.

modalities in T2DM, even though changes in a number of the key variables did not reach statistical difference. Total body fat mass and android fat percentage were reduced after 12 and 24 weeks of football training

Football training and T2DM compared with before IP (Fig. 4(a,b)), which may be explained, in part, by the intense work performed during training sessions with about half the session performed at intensities above the pace of jogging and with HR being higher than 80% of individual maximal heart rate for as much as 27 min on average in each session. High-intensity interval training has been shown to be equally effective as longer periods of moderateintensity training in regard to reduction in total body fat mass (Schjerve et al., 2008), and has been reported to elicit preferential reductions in intra-abdominal adipose tissue, which may be associated with improved glycemic control (Mourier et al., 1997; Irving et al., 2008). The present study suggests that football training is an effective training modality for reducing total adipose tissue mass and android fat percentage in a population of overweight (BMI > 30) men with T2DM. These changes in body composition may impact insulin sensitivity and glycemic control since changes in visceral adipose tissue are positively related to changes in HOMA-IR (Okita et al., 2013). In CG, a decrease in leg lean mass and leg bone mass (Table 1) was observed during the study, which does not appear to have been caused by a reduction in the daily activity during the intervention period (Schmidt et al., 2013). However, pedometer measures were performed only at selected time points and may not be specific for the total intervention period, and the self-reported activity may have been overestimated by the physical activity scale questionnaire. VO2peak was increased by 10% after 12 weeks, with no further rise during the following 12 weeks, which is similar to what has been reported for aerobic training of T2DM subjects in a meta-analysis (Boule et al., 2003; Fig. 3). Also, a newly reported study investigating the effects of 16 weeks of training involving 1 h of interval walking five times a week in T2DM patients showed a 16% increase in VO2max, which was not evident with continuous moderate-intensity walking at the same mean caloric expenditure (Karstoft et al., 2013). Thus, the application of intense exercise appears to be efficient at improving cardiovascular fitness in T2DM subjects (Sigal et al., 2013), and this notion is supported by evidence from studies using high-intensity interval bicycle (Lithell et al., 1985) or treadmill (Mitranun et al., 2014) training. In accordance, we recently found improved cardiac function and marked reductions in blood pressure after a period of football training in T2DM patients (Schmidt et al., 2013). When expressing peak oxygen uptake per unit body mass (Table 1), the observed changes after 24 weeks were of similar magnitude, which is probably primarily due to changes in the cardiovascular system, underlining the effectiveness of football training in improving health in a group of type 2 diabetic subjects. Thus, an increasing level of cardiorespiratory fitness of approximately 5 mL/min/ is associated with significant reductions in cardiovascular

and overall mortality of 39% to 70% over 15 to 20 years of follow-up in subjects with diabetes (Church et al., 2005). It was a striking finding that the CS activity was unaltered, and the expression of CS was lower after the period of football training, which has been shown in untrained male subjects to lead to marked increases in CS activity (Krustrup et al., 2010). Before IP, the CS activity was low and at a similar level to that of untrained subjects (Duscha et al., 2012), probably reflecting the fact that the T2DM subjects had an inactive lifestyle taking less than 5000 steps a day. The reason for the lack of response in muscle CS in the present study is unclear, and generally, the expression of most of the muscle proteins analyzed, with the exception of Akt-2, was unchanged after 24 weeks compared with before IP. Furthermore, mean muscle fiber size was unaltered in FG after 24 weeks of football training, which is in contrast to findings of an increase in mean fiber area after 12 weeks of recreational football training in healthy untrained middle-aged males (Randers et al., 2010a). Apparently, the muscular training response (e.g., muscle growth) is inhibited in T2DM patients and stronger stimuli are needed to create muscle changes. FGF-21 has been suggested as a novel biomarker of metabolic regulation. Elevated levels of FGF-21 are linked with enhanced muscle glucose uptake and inhibition of lipolysis in adipocytes, which has been speculated to be a compensatory mechanism to counteract the metabolic abnormalities imposed by T2DM and obesity (Bergmann & Sypniewska, 2013). We applied an ANCOVA model to the data analysis of FGF-21 correcting for BMI, total body fat mass, fasting plasma glucose, fasting plasma insulin, blood lipids and cholesterol, VO2max, and baseline levels in FGF-21, and found that FGF-21 was not changed during the 24 weeks of football training. Recent data have showed a large range of serum FGF-21 levels depending on the subtype and severity of diabetes (Xiao et al., 2012), and this may reflect the fact that T2DM is not an uniform condition, but comprises many different pathophysiological subtypes (Steffensen et al., 2012). The intervention groups were not matched for FGF-21 at baseline, and this may have contributed to excess biological variation making a change in FGF-21 undetectable in the present study. As such, factors not included in the statistical model could have influenced our findings, and a training effect on FGF-21 in T2DM could not be established in the present study The present study demonstrated that the T2DM patients were able to cope with the high loads during the football training, but also that the demands of the training varied significantly. Thus, during a training session, blood lactate levels ranged from 2.3 to 12.8 mmol/L (Fig. 2(c)) percentage of effective playing time spent at intensities > 85% HRmax ranged from 4% to 95% (Fig. 2(a)), and distance covered above the pace of

53

Andersen et al. jogging ranged from 1.5 to 3.6 km (Fig. 2(b)). However, all subjects reported the overall training load as being strenuous; the average HR during training was > 70% HRmax for all subjects and most subjects had a mean heart rate > 80% HRmax, which is not often well tolerated by untrained subjects. An increase in intermittent endurance capacity was observed in all subjects (Schmidt et al., 2013) and VO2peak increased in 11 out of 12 subjects (Fig. 3). The subject with no change in VO2peak had a marked lowering of HbA1c (0.4%), a reduction in total body fat mass (3 kg), and a lowering of android fat percentage (5%), and thus also benefitted from the training. No relationship between initial fitness status and change in VO2peak during IP was observed. Apparently, the football training effectively affected all the participants irrespective of initial physical capacity and prior experience of playing football, and football training conducted as small-sided games appears to be an exercise modality, which ameliorates a wide range of physiological parameters in men with T2DM. A number of limitations apply to the present study. The study was not randomized and the group assignment may have influenced the results. In addition, the number of subjects in each of the intervention groups was low, which may have increased the risk of conducting statistical type 2 errors, and may be the reason why changes in a number of the key variables for glycemic control were not significant. As such, the general direction of the findings in the present study needs to be confirmed by future larger and randomized studies In summary, 24 weeks of recreational football training improved VO2peak, and lowered total body and android fat mass in middle-aged men with T2DM. Additionally, indications of a positive impact of football training on glycemic control were observed. These changes may be associated with reduced long-term morbidity and football may be an attractive contribution to the treatment of T2DM.

Perspectives For patients with T2DM, adherence to exercise prescription is of paramount importance. In the present study, a high training adherence (∼75%) was observed. Additionally, it is notable that recreational football requires little preparation and no prior experience, and can be played anywhere with a limited number of participants. Thus, recreational football may be easily accessible, attractive, and not time consuming for T2DM subjects. In addition to a wide range of physiological benefits, football has a significant psychological-social component, which is important for lifelong engagement in physical exercise training. A topic thoroughly covered in the companion paper by Nielsen et al. (2014). Improving health status through regular exercise lowers the need for pharmacological treatment and is of marked socioeconomic importance. We therefore recommend that recreational football should be a part of an integrated approach alongside diet intervention and drug therapy in the treatment and prevention of T2DM. Key words: Soccer, high-intensity training, oxidative capacity, health, team sport, FGF-21.

Acknowledgements We sincerely thank the participants in the study for their efforts. Also, the technical and practical assistance of Joshua Horton, Jonathan Brix, Marie Von Hagman and Allan Smedemark is greatly appreciated. Conflicts of interest: None of the authors have any conflict of interest in the present results.

Funding The study was supported by the FIFA Medical Assessment and Research Centre (F-MARC) and Nordeafonden, Denmark.

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