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Repeated Prolonged Exercise Decreases Maximal Fat Oxidation in Older Men


Medicine & Science in Sports & Exercise: February 2017 - Volume 49 - Issue 2 - p 308–316
doi: 10.1249/MSS.0000000000001107
Applied Sciences

Introduction/Purpose Fat metabolism and muscle adaptation was investigated in six older trained men (age, 61 ± 4 yr; V˙O2max, 48 ± 2 mL·kg−1·min−1) after repeated prolonged exercise).

Methods A distance of 2706 km (1681 miles) cycling was performed over 14 d, and a blood sample and a muscle biopsy were obtained at rest after an overnight fast before and 30 h after the completion of the cycling. V˙O2max and maximal fat oxidation were measured using incremental exercise tests. HR was continuously sampled during cycling to estimate exercise intensity.

Results The daily duration of exercise was 10 h and 31 ± 37 min, and the mean intensity was 53% ± 1% of V˙O2max. Body weight remained unchanged. V˙O2max and maximal fat oxidation rate decreased by 6% ± 2% (P = 0.04) and 32% ± 8% (P < 0.01), respectively. The exercise intensity that elicits maximal fat oxidation was not significantly decreased. Plasma free fatty acid (FA) concentration decreased (P < 0.002) from 500 ± 77 μmol·L−1 to 160 ± 38 μmol·L−1. Plasma glucose concentration as well as muscle glycogen, myoglobin, and triacylglycerol content remained unchanged. Muscle citrate synthase and ß-hydroxy-acyl-CoA-dehydrogenase activities were unchanged, but the protein expression of HKII, GLUT4, and adipose triacylglycerol lipase were significantly increased.

Conclusions Overall, the decreased maximal fat oxidation was probably due to lower exogenous plasma fatty acid availability and the muscle adaptation pattern indicates an increased glucose transport capacity and an increased muscle lipolysis capacity supporting an increased contribution of exogenous glucose and endogenous fat during exercise.

Supplemental digital content is available in the text.

1Xlab, Center for Healthy Aging, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, DENMARK; 2Department of Bioscience, Zoophysiology, Aarhus University, Aarhus, DENMARK; 3Department of Cardiology, University Hospital of Bispebjerg, Copenhagen, DENMARK; 4Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, SWEDEN; 5Department of Anaesthesia and Intensive Care, Karolinska University Hospital, Huddinge, Stockholm, SWEDEN; and 6Institute of Sports Medicine Copenhagen, Bispebjerg Hospital, Copenhagen, DENMARK

Address for correspondence: Thomas Morville, M.D., Department of Biomedical Science, Faculty of Health Science, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; E-mail:

Submitted for publication June 2016.

Accepted for publication September 2016.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (

In the last decade, ultraendurance exercise and very prolonged exercise have become increasingly popular and this has led to research attempting to enhance our understanding of the mechanisms and limitation for these activities. A well-known effect of endurance training is an increase in whole-body fat oxidation, if exercise is performed at the same absolute and relative intensity (15,26). Indeed maximal fat oxidation and the exercise intensity where this occurs are increased with training (24,25). In line with this, we have shown increased fat oxidation during both low and moderate-intensity cycling exercise in the fed condition after more than 100 h of continuous mixed ultraendurance exercise (13). Furthermore, Slivka and colleagues (38) demonstrated that 19 d of 5–6 h continuous moderate intensity road cycling increased fat oxidation during moderate intensity exercise in young well-trained men. There is, however, limited information about the effect of repeated prolonged exercise on fat metabolism, maximal fat oxidation, and muscle adaptation in older trained individuals. In general, there is agreement that older adults similarly to younger adults respond to endurance training of sufficient intensity with increased maximal oxygen uptake as well as mitochondrial adaptations with increased mitochondrial capacity (16,20,22,34).

The influence of age on fat oxidation during exercise has been investigated in many studies, but unfortunately, the evidence is contradicting showing both increased, unchanged or reduced fat oxidation with age (reviewed in (23,39)). Furthermore, after endurance training, both young and older individuals have an increase in fat oxidation during exercise although the increase is attenuated with age (22,36). It is not clear whether increased fat oxidation is mediated through changes in substrate recruitment and availability, and/or adaptation in fat and carbohydrate oxidative pathways in skeletal muscle after repeated prolonged exercise.

Based on this, we studied maximal fat oxidation and factors regulating fat oxidation in skeletal muscle in six older male recreationally trained cyclists after repeated prolonged exercise. We hypothesized that an increased maximal fat oxidation and an increased muscle fat oxidative capacity comparable to that observed in young trained adults would be found after the repeated prolonged exercise.

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Study Design

The design of this study has been published in detail elsewhere (31). In brief, six male recreational cyclists (61.3 ± 3.8 yr, V˙O2max 48 ± 2 mL·kg−1·min−1) participated in the study, and all subjects were recreational skilled cyclists. On their own accord, to win a bet (not initiated by any of the authors), the subjects organized a 14-d cycle trip with a total distance of 2706 km (1681 miles) from the Town Hall Square in Copenhagen, Denmark, to North Cape in Norway, as far north as possible on the European continent (Fig. 1, (31)). On this trip, the subjects' own support team handled all the practical logistics, providing transport of extra gear and maintenance of bikes and supplying, cooking, and preparing all food and beverages before, during, and after cycling. Dietary intake was assessed using a self-reported diet record in the week before departure (three weekdays and one weekend day) and was registered by the scientific staff during three periods under cycling (days 2–3, days 5 and 7, and days 12–13). In all cases, all foods and drinks were carefully measured and weighed to the nearest gram. All dietary records were processed appropriate software (Dankost 3000; Dansk Catering Service, Gentofte, Denmark). Two to 5 d before leaving and again 28–33 h after arrival at North Cape, the subjects were tested under standardized conditions in the morning after an overnight fast. All subjects were tested using the same equipment and protocols on both occasions. A month before departure, five of six subjects participated in an optional test day to be familiarized with the test procedures. One subject was unavailable due to job constraints. The tests after the 14 d of cycling were performed in a hostel at North Cape, and therefore, it was not possible to perform a dual-energy X-ray absorptiometry scan. The sampling of blood and muscle tissue was done exactly as before the cycling in Copenhagen, and efforts were made to do the tests at the same time of day as well. In the day before the test day, subjects were asked to consume their habitual diet and to refrain from participating in strenuous or prolonged exercise.



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Ethical Approval

The subjects were informed about the possible risks and discomfort involved before written consent to participate was obtained. The study was performed according to the Declaration of Helsinki and was approved by the Science Ethical Committee of the Copenhagen Region (H3-2011-008) and registered at (NCT02353624).

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Experimental Conditions

Subjects arrived in the morning overnight fasted, and after a 15-min rest, a venous blood sample was collected from an antecubital vein. After this, a needle muscle biopsy was obtained with suction from vastus lateralis muscle.

After this, two graded bicycle ergometer (Jaeger ER 800; Würzburg, Germany) exercise tests were performed starting with a Fatmax protocol where the initial workload was 60 W for 5 min and then progressively increased by 35 W every 3 min until the RER was above 1.0. After a 5-min break, a maximal oxygen uptake (V˙O2max) protocol was started with a 100-W workload for 3 min followed by increments of 40 W·min−1 until exhaustion. Attainment of maximal oxygen uptake was ascertained by the following criteria: a leveling off of V˙O2 despite an increase in power output, a respiratory exchange ratio exceeding 1.15, and achievement of estimated maximal HR. During exercise, respiratory gases were sampled with an online system (CosMed; Quark b2, Rome, Italy).

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Analytical Procedures

Blood analysis

Blood was transferred into tubes containing 0.3 mol·L−1 EDTA (10 μL·mL−1 blood) and immediately centrifuged at 4°C for 10 min at 23,000g. The plasma was stored at −80°C until analysis. Plasma glucose was analyzed on an automatic analyser Cobas 6000 c 501 (Roche, Glostrup, Denmark). Concentrations of plasma insulin were determined by a commercially available ELISA kits (Dako, Glostrup, Denmark) and along with plasma cortisol (ELISA reference: DE1887; Demeditec Diagnostics GmbH), they were read on a Thermo Scientific Multiskan FC (Thermo Fisher Scientific Oy, Vantaa, Finland). Plasma free fatty acid (FA) concentration was measured using a Wako NEFA-C test kit (Wako Chemical, Neuss, Germany) on an automated analyser Cobas 6000 c 501 (Roche, Glostrup, Denmark). The plasma concentration of adiponectin was measured by a human radioimmunoassay kit (EMD Millipore.; cat. no. HADP-61HK, Billerica, MA).

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Skeletal muscle analysis

The muscle tissue was frozen within 10–15 s of sampling. Before freezing, a section of the samples was cut off, mounted in embedding medium and frozen in isopentane, cooled to its freezing point. Both parts of the biopsy were stored at −80°C until further analysis. Before biochemical analysis, muscle biopsy samples were freeze-dried and dissected free of connective tissue, visible fat, and blood using a stereomicroscope. Muscle capillary density was analyzed and quantified by immunohistochemistry as described by Qu et al. (27). The maximal activity of the enzymes HAD and CS was determined fluorometrically as described previously (4). The muscle glycogen and triacylglycerol content were measured enzymatically as described previously (11).

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The concentration of myoglobin (Mb) was measured spectrophotometrically using a modified version of the method by Reynfarje (28). 19.25 mL buffer per gram of tissue (0.04 M potassium phosphate, pH 6.6) was added to muscle biopsies weighing approximately 30 mg, and samples were homogenized for 5 × 1 min with 1-min intervals in between on ice using a TissueLyser LT homogenizer (Quiagen, Retsch, Haan, Germany). Samples were centrifuged for 50 min at 15,000g at 4°C, and the supernatant was equilibrated with pure CO gas for 3 min. A pinch of dithionite was then added, and the sample was equilibrated with CO for one more minute. Finally, the absorbance was recorded at 538 and 568 nm using a HP 8543UV-visible diode array spectrophotometer, and the concentration of Mb was calculated using the formula:

where OD is optical density. Longer CO equilibration times made no changes to the Mb concentration measurements and samples were therefore considered as being 100% CO saturated.

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Western blotting

For the Western blotting, approximately 20 mg (w.w.) of the biopsies were homogenized in 350 μL cold buffer added protease and phosphates inhibitors (50 mM Tris pH 8.0, 150 NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 2.5 mM PMSF, 20 mM β-glycerophosphate, 10 mM pyrophosphate, 2 mM Sodium Ortovanadate, including mini EDTA protease inhibitor tablet according to the instructions of the manufacturer (Roche Diagnostics, Mannheim, Germany). Homogenization was done at 30 Hz for 2 × 2 min at −20°C in a TissueLyser (Qiagen Retsch, Haan, Germany), or until the sample was completely dissolved. Thereafter, the homogenate was sonicated for 2 × 5 s. Protein concentration was measured by bicinchoninic acid assay (Pierce, Rockford, IL) in triplicate, and a maximal coefficient of variation of 5% between replicates was accepted.

An equal amount of protein (20 μg) was heated to 95°C for 10 min and electrophoresed in either 12.5% or 4%–15% polyacrylamide sodium dodecyl sulphate gels (26 well 2-amino-2-(hydroxymethyl)-1,3-propandiol (Tris)-HCl precast gel, Criterion, Bio-rad, Copenhagen, Denmark) and electrotransferred to a PVDF membrane (Bio-Rad, Copenhagen, Denmark). To check for even transfer throughout the membranes, a homogenate was loaded on each gel on several spots dispersed all over the gel. This homogenate was made from a mix of biopsies from two participants that were matched by age, V˙O2max and fat-free mass and handled as the other samples. It is difficult to find a reference gene that is not affected by age or by fitness level, and therefore, we chose not to stain for a reference protein on the membranes. However, to control equal protein loading and also transfer efficacy from gels to membranes, all our gels were stained with Coomassie Blue (Pierce). The Coomassie-stained gels were subsequently imaged using a LAS 4000 image analyser (GE Healthcare, Little Chalfont, UK). The images were used to quantify the total protein stain of each lane, and this was used as a measure of equal loading. A variation of 10% of the average intensity between the lanes was accepted.

The membranes were blocked for 0.5 to 1.5 h at room temperature with either skimmed milk (Merck, Darmstadt, Germany) or bovine serum albumin (BSA, Fraction V Modified Cohn) diluted in Tris-buffered saline (10 mM Tris Base, 150 mM NaCl, pH 7.4) ± 0.05% Tween 20. The membranes were then incubated with the primary antibody overnight at 4°C. The primary antibodies were anti-adipose triacylglycerol lipase (ATGL) (cat. no. 2138; Cell Signaling Technology, Beverly, MA), anti-CD36 (cat no. ab17044; Abcam, Cambridge, UK), anti-FABPpm (GOT2, Cat nr. Ab93928; Abcam), anti-GLUT4 (cat no. PA1-1065; Thermo Scientific, Rockford, IL), anti-Hexokinase II (cat. no. 2867; Cell Signaling Technology), anti–hormone sensitive lipase (HSL) (G7) (cat. no. sc-74489, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), anti-LPL (H53) (cat. no. sc-32885, Santa Cruz Biotechnology, Inc.), and anti–vascular endothelial growth factor (VEGF) (cat. no. J806; Santa Cruz Biotechnology, Inc.). The membranes were washed three times in Tris-buffered saline ± 0.05% Tween 20. Secondary antibodies were goat antirabbit horseradish peroxidase conjugated and goat antimouse horseradish peroxidase conjugated (Dako). Blots were developed in ECL detection reagents (GE Healthcare), and the chemiluminescence emitted from immune-complexes was visualized with a LAS 4000 image analyzer (GE Healthcare). The images of the membranes and Coomassie-stained gels were quantified by ImageQuant TL software version 7.0 (GE Healthcare).

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Mass spectrometry

Sphingolipids and ceramides were analyzed using a Waters Acquity UPLC system (Milford, MA) coupled to Thermo TSQ Quantum Ultra mass spectrometer (Waltham, MA), as described before (5). Briefly, muscle was cleaned from fat and connecting tissue and subjected to homogenization. Sphingolipids and ceramides were extracted with internal standard containing D-erythro-sphingosine-1-phosphate (C17 base), D-erythro-sphingosine (C17 base), N-heptadecanoyl-D-erythro-sphingosine and N-nervonoyl-D-erythro-sphingosine using an extraction solution of ethyl acetate/isopropanol/water (60:35:5, v/v/v). Samples were centrifuged, the supernatant was saved, and the solvent evaporated under a stream of nitrogen at room temperature.

Standards of sphingosine, sphingosine-1-phosphate, sphinganinge, N-myristoyl-D-erythro-sphingosine (C14-cer), N-palmitoyl-D-erythro-sphingosine (C16-cer), N-oleoyl-D-erythro-sphingosine (C18:1-cer), N-stearoyl-D-erythro-sphingosine (C18-cer), N-arachidoyl-D-erythro-sphingosine (C20-cer), N-behenoyl-D-erythro-sphingosine (C22-cer), N-nervonoyl-D-erythro-sphingosine (C24:1-cer), and N-lignoceroyl-D-erythro-sphingosine (C24-cer) were purchased from Avanti Polar Lipids (Alabaster, AL). UPLC-MS/MS analysis was accomplished on a Acquity UPLC BEH C8 column (Waters, 2.1 × 150 mm, 1.7 μm) using a mobile phase of buffer A 99% water, 1% MeOH, 2 mM ammonium formate, 0.1% formic acid and buffer B 99% MeOH, 1% water, 1 mM ammonium formate, 0.1% formic acid. The mass spectrometer was run in positive ionization mode using SRM using an 11-point standard curve (5).

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Whole-body fat oxidation was calculated from the respiratory exchange ratio during the last 60 s of each exercise step in the graded exercise test using standard indirect calorimetry equations (10). As previously described, maximal whole-body fat oxidation and the exercise intensity, which elicits this, is estimated based on mathematical modelling of each subject's data (25).

To estimate exercise intensity during the 14 d of cycling, individual relationships between HR and oxygen uptake were established from the incremental maximal oxygen uptake tests before the 14 d. For all 14 d, the relative exercise intensity was calculated for every 10-min period, and from this, we calculated average daily exercise intensity and the average daily duration of exercise over 60% of V˙O2max. The calculated exercise intensity cannot account for the drift in HR that will occur as a consequence of the very prolonged exercise. Therefore, the calculated exercise intensity is a minimum value.

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Statistical analysis

The statistics were calculated using SigmaPlot ver. 11.0 (Systat Software Inc., San Jose, CA) and Graph Pad Prism, version 5.01 (GraphPad Software Inc., La Jolla, CA). To assess changes before and after the 14 d of cycling, a two-tailed paired t test was used. Nonparametric data were tabulated as medians with 25%–75% percentiles, and differences were evaluated with ranked statistical tests (Mann–Whitney for pre-post measures and Shapiro–Wilks for repeated measures). Results are given as mean ± SEM, if not otherwise stated. In all cases, P < 0.05 was taken as the level of significance in a two-tailed test. A P value of 0.05–0.1 was defined as trend because significance could not be established due to small sample size.

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Study characteristics

The characteristics of the cycling trip have been published previously (31). In summary, all six subjects completed the full distance of 2706 km (1681 miles) in 14 d with an average daily distance of 193 ± 10 km·d−1 (range, 167–235 km·d−1). Daily exercise time was 10 h and 31 ± 37 min·d−1 with an exercise intensity of 53.1% ± 1.1% of V˙O2max and approximately 198 ± 58 min·d−1 exercise intensity above 60% of V˙O2max. After cycling, maximal oxygen uptake was reduced (48 ± 2 mL to 45 ± 2 mL O2·min−1·kg−1, P < 0.04). As previously reported, we observed a 2.2 ± 0.7 kg loss of fat mass and 2.5 ± 0.6 kg gain of lean mass after 8 d of cycling using isotope dilution technique (31). Total energy consumption doubled during cycling (110% ± 4%) due to an increase in all macronutrients (Table 1). In terms of dietary macronutrient composition, the relative contribution of carbohydrate increased (P < 0.001) at the expense of dietary protein (P < 0.01) and, to some degree, relative fat intake (P = 0.08) (Table 1).

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Fat oxidation and blood analysis

Over the course of cycling there was robust 32% ± 4% decrease in maximal whole body fat oxidation from 0.63 to 0.43 g·min−1 (Fig. 2A). Furthermore, there was a tendency toward a decrease in the exercise intensity that elicits maximal fat oxidation from 59% ± 5% to 54% ± 2% (Fig. 2B). The power outputs at which FatMax occurred tended to be lower after cycling with a decrease from 231 to 198 W (P = 0.06). Blood analysis showed that there was a large decrease (P < 0.01) in fasting plasma FA concentration after cycling, whereas both plasma lactate and glucose concentrations at rest remained unchanged (Table 2). Plasma insulin was higher after cycling leading to a subsequent trend (P = 0.06) toward an increase in HOMA-IR (homeostatic model assessment-insulin resistance) (Table 2). Plasma adiponectin as well as cortisol concentrations were unchanged (Table 2). There was no correlation between the concentrations of insulin and FA.



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Skeletal muscle morphology and characteristics

After cycling skeletal muscle capillarization, expressed both per area skeletal muscle and per capillary, as well as VEGF content and muscle Mb were unchanged (Table 3). In terms of sarcolemmal substrate transport; GLUT4 protein content was increased (Fig. 3A, P < 0.01) but FA transporters CD36 and FABPpm (GOT2) were not influenced by cycling (Fig. 3B). Muscle glycogen and intramuscular triacylglycerol and CS and HAD activities remained unchanged (Table 3). HK II (P < 0.02) protein content was increased (Fig. 3A) and muscle ATGL content was higher (Fig. 3B, P < 0.05), and there was a trend towards an increase in lipoprotein lipase (LPL) content (Fig. 3C, P = 0.06). Nevertheless, HSL content was not significantly changed (Fig. 3C) (see Figure, Supplemental Digital Content 1, representative blots for GLUT4, HKII, LPL, FABPpm, CD36, ATGL, HSL, and VEGF,









There were no changes in the content of diglyceride acyltransferase (DGAT)1 and DGAT2 or the content of perilipin 2, 3, and 5 in muscle after cycling (Table 3), and the content of sphingosine and ceramide derivatives were also unchanged except from a decrease (P = 0.035) in C24:1-ceramide (see Table, Supplemental Digital Content 2, content of ceramide and sphingosine derivatives from homogenized muscle,

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In the present study, our hypothesis was refuted as the major finding was not an increased but rather a decreased maximal fat oxidation after 14 d of prolonged repeated exercise in older men. In addition, the CS and HAD activity remained unchanged in contrast to our hypothesis. Interestingly, we observed an increased GLUT4 and HKII content and a higher ATGL muscle content supporting an increased capacity to utilize exogenous carbohydrate and maintain an endogenous FA supply. Finally, the plasma FA concentration at rest was surprisingly decreased by a factor of 3, which may explain the lack of an increase in fat oxidative capacity and a decreased maximal fat oxidation.

In a recent study of young trained men exposed to 3211 km of cycling in 19 d, where the exercise was comparable to that performed in the present study, increased fat oxidation during exercise was observed over a range of exercise intensities after the intervention (38). This is clearly in contrast to the present observation of a marked decrease in maximal fat oxidation in older trained men after 14 d cycling. In this field study, we were limited to sampling blood and muscle before exercise and do not have direct measurements of endogenous and exogenous substrate utilization during exercise. Substrate partitioning at maximal fat oxidation is not well characterized in the literature, but in two studies, the contribution of endogenous and exogenous fat was approximately 50% each at exercise intensities close to that considered to elicit maximal fat oxidation in young men (30,41). During whole-body exercise, plasma FA delivery is one of the major determinants of FA oxidation (29,30). We observed a marked decrease in plasma FA concentration at rest, and this contrasts findings of unchanged plasma FA concentration at rest in older subjects after endurance training (36). We do not have a good explanation for the observed lower plasma FA at rest, but we observed higher plasma insulin concentration at rest (31), and this may have contributed to the lower plasma FA by an inhibition of lipolysis in the adipose tissue. The observed discrepancy in FA availability and insulin levels between the present study and studies of endurance training in elderly may be explained by the amount of exercise as well as the balance between rest and recovery. In addition, we speculate that the massive amount of exercise could have induced desensitization to catecholamine stimulation induced by a decreased ß-adrenoceptor response and/or decreased receptor number (19,43), which could have reduced the lipolysis rate and/or the blood flow in the adipose tissue also leading to a decreased plasma FA concentration. Catecholamines were not measured; however, indications of the level of stress were reflected in the nonsignificant increase in cortisol concentrations in five of the six participants (28% increase pre vs post, n = 6). Along with elevated insulin concentrations, these cortisol levels could result in decreased lipolysis.

We acknowledge that substrate metabolism and storage are dependent on nutritional status, and such factors could influence the results presented in the study. We made a large effort to observe and register foods and drinks consumed during cycling, as previously published (31), but a limitation is the lack of dietary control the days before testing. We therefore do not have control whether possible overeating occurred and that subjects thus were in positive energy balance on the day before posttesting, but some indications suggest that they were not. Given the massive daily amount of carbohydrate consumed during cycling and as a consequence of a potential positive energy balance (prior to post testing), an increase in glycogen resynthesis and storage would be expected, but muscle glycogen storage remained at similar levels after cycling as compared with baseline. The maximal storage capacity of muscle glycogen in man is 15 g·kg−1, and at such levels, lipogenesis occurs (1), but we do not suspect the subjects to have reached a saturation in glycogen storage, given the levels of muscle glycogen presented here. Rather, the increase in exogenous supply carbohydrate suggests a larger turnover of carbohydrates. As carbohydrate balance is relatively precisely controlled, the increase in the food quotient should increase respiratory quotient (9) and most likely give rise to a lower FatMax.

Another implication for the reduced glycogen storage could be the increase in alcohol intake over cycling. However, the amount of alcohol ingested during this study is still fairly low, despite the doubling of intake. In far larger doses of alcohol than in the present study, glycogen resynthesis after prolonged exercise is affected when alcohol displaces CHO, but not when given in combination with adequate dietary CHO (6) as in the present study.

We observed a very distinct increase in the muscle GLUT4 and HKII levels after the 14 d, which is in line with prior training studies (7,42) and implies an induced increased high glucose transport capacity, which support a high exogenous carbohydrate utilization during exercise.

Muscle triacylglycerol storage was also unchanged after 14 d of cycling, which is consistent with the lack of changes in the content of DGAT1 and DGAT2 and the content of lipid droplet proteins perilipin 2, 3, and 5. However, we observed higher muscle LPL (P = 0.06) and ATGL content after 14 d of cycling, which is line with former studies demonstrating that regular endurance training induced a higher muscle ATGL content (2) and muscle LPL content (17,35). In the present study, the increased LPL content was not accompanied by increases in the level of the muscle membrane FA transporters CD36 and FABPpm, and we speculate that the increased LPL content is a reflection of a rather massive exogenous fuel recruitment required to complete more than 10 h of cycling exercise per day during 14 d. The increased ATGL content is not readily explained, but could be due to an increased IMTG breakdown during rest to accomplish faster muscle glycogen replenishment (18).

Interestingly, we observed an increase in HOMA, and thus decreased insulin sensitivity, caused by an increase in circulating insulin (31). Ceramide and ceramide subspecies are bioactive lipids that together with diacylglycerol are thought to be mediators of insulin resistance (3,14). However, the lack of change in muscle ceramide content despite a small increase in insulin resistance is fully in line with our prior work, where no change in ceramide content was observed despite major changes in insulin sensitivity (37). The current observation of unchanged ceramide and sphingosine content is in agreement with former studies showing no difference in ceramide content between trained and untrained subjects (12) or after 12 wk endurance training (8). The observation of an increased HOMA-IR despite increased muscle GLUT4 and HKII content and lowered plasma FA concentration is somewhat surprising, but may have been stimulated by the very high carbohydrate consumption over the 14 d.

In the present study, muscle CS and HAD activities remained unchanged after 14 d, whereas Slivka and colleagues (38) observed a borderline increased (P = 0.055) CS activity and unchanged HAD activity after 19 d of cycling in young men. Our finding is unexpected because mitochondrial adaptations after such activity has been shown to present also in elderly individuals (16). Although the exact mechanisms that control the partitioning of fat and carbohydrate for oxidation are not fully revealed (32,33), it is probable that an increased CS activity through a higher TCA flux will contribute to a higher fat oxidation during exercise and thus, at least in part, explain the observed difference in fat oxidation after repeated prolonged exercise between the present and the study by Slivka and colleagues (38). Although there was only a trend (P = 0.07) toward a decrease in the exercise intensity at which maximal fat oxidation is elicited as well as a trend (P = 0.06) toward lower power outputs, the present data and the observation of a decreased V˙O2max suggest a leftward and downward shift in the bell-shaped fat oxidation curve consistent with the difference between trained and untrained subjects (25).

As reported in a prior publication, we observed an unexpected 6% ± 2% decrease in maximal oxygen uptake after 14 d of prolonged cycling exercise in older men (31). The unchanged CS and HAD activities reported here imply that mitochondrial capacity cannot explain this observation.

In the present study, 2 wk of prolonged exercise did not increase capillarization, and in line with this, muscle VEGF content remained unchanged. Moreover, muscle Mb content remained unchanged after prolonged exercise, which is in line with earlier studies in human muscle showing unchanged Mb content after training (21,40). Overall, this indicates that the repeated prolonged exercise did not increase local blood perfusion or oxygen transport capacity in older men. However, it is possible that these older men are indeed so well trained that their inherent capacity to respond further to the training stimulus is limited. Our data set does not provide a mechanistic explanation for the observed decrease in maximal oxygen uptake. However, blood hemoglobin concentration and total blood hemoglobin mass were not measured in this study and although we find it unlikely, it cannot be excluded that a decreased blood oxygen carrying capacity could account for the observed lower maximal oxygen uptake. Another possible and more likely explanation is an exercise-induced desensitization to catecholamines caused by a decreased ß-adrenoceptor response and/or receptor number (19,43), which would decrease cardiac output and possibly venous return during maximal exercise.

In summary, maximal fat oxidation and maximal oxygen uptake decreases after repeated prolonged exercise in older trained men. The observed attenuated plasma FA concentration at rest after the intervention suggests a decreased delivery of FA during exercise thus limiting the maximal fat oxidation through a decreased contribution from exogenous fat. In line with this, the observed increased muscle glucose transport capacity supports an increased utilization of exogenous carbohydrates. Although the older men were able to complete the cycling in 14 d, the counterintuitive observations, decreased maximal fat oxidation and maximal oxygen uptake, imply that the exercise load was probably close to the sustainable maximal workload for these older individuals.

Materials for the bicycle trip were supported by Maribo Medico A/S, Toms Gruppen A/S, and Orkla Health. B. T. received a travel award from the Karolinska Institutet-Mayo Foundation and Karolinska Institutet Fonder. This work was supported by the Lundbeck Foundation (grant R0-A975 to A. F.) and by the Danish Council for Independent Research, Natural Sciences (grant 10–084565 to A. F.).

The skilled technical assistance of Thomas Nyegaard Beck, Regitze Kraunsøe, Jeppe Bach, and Xuan-Mai T. Persson and Jaime Gransee from Mayo Clinic Metabolomic Research Core are gratefully acknowledged.

The authors have no conflict of interest in relation to the present scientific paper. The results do not constitute endorsement by American College of Sports Medicine and they are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.

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