Journal of Applied Physiology

Intensity-controlled treadmill running in mice: cardiac and skeletal muscle hypertrophy

Ole Johan Kemi, Jan P. Loennechen, Ulrik Wisløff, Øyvind Ellingsen


Whereas novel pathways of pathological heart enlargement have been unveiled by thoracic aorta constriction in genetically modified mice, the molecular mechanisms of adaptive cardiac hypertrophy remain virtually unexplored and call for an effective and well-characterized model of physiological mechanical loading. Experimental procedures of maximal oxygen consumption (V˙o 2 max) and intensity-controlled treadmill running were established in 40 female and 36 male C57BL/6J mice. An inclination-dependent V˙o 2 maxwith 0.98 test-retest correlation was found at 25° treadmill grade. Running for 2 h/day, 5 days/wk, in intervals of 8 min at 85–90% of V˙o 2 max and 2 min at 50% (adjusted to weekly V˙o 2 max testing) increasedV˙o 2 max to a plateau 49% above sedentary females and 29% in males. Running economy improved in both sexes, and echocardiography indicated significantly increased left ventricle posterior wall thickness. Ventricular weights increased by 19–29 and 12–17% in females and males, respectively, whereas cardiomyocyte dimensions increased by 20–32, and 17–23% in females and males, respectively; skeletal muscle mass increased by 12–18%. Thus the model mimics human responses to exercise and can be used in future studies of molecular mechanisms underlying these adaptations.

  • maximal oxygen uptake
  • work economy
  • respiratory exchange ratio
  • cardiomyocyte
  • allometric scaling

several novel signaling pathways of pathological cardiac hypertrophy have been unveiled in the powerful model of mechanical overloading by thoracic aorta constriction in genetically modified mice (8, 22). Thus far, the molecular mechanisms of adaptive cardiac hypertrophy in response to physiological stimuli remain virtually unexplored because of a paucity of effective and well-controlled experimental models in this species. Ideally, the outcome should mimic typical results of exercise training in humans, e.g., increased maximal oxygen uptake (V˙o 2 max) and work economy (21). In healthy individuals, increased exercise capacity is dependent on functional and structural adaptations in skeletal muscle, blood vessels, and heart. Cardiac adaptation includes increased ventricular chamber volumes and weights, cardiomyocyte hypertrophy, increased cardiac output, and improved contractile function measured in isolated cardiomyocytes and in the intact heart (2, 3).

At present, there are few well-controlled endurance training studies in mice. Training is usually carried out as voluntary exercise regimens, with little control of exercise intensity and the individual's fitness level. Voluntary wheel running is most frequently used.V˙o 2 max has been shown to increase by 12–30% after a 7- to 12-wk period of free wheel running (11, 40). The latter study also demonstrated a 6% decreased respiratory exchange ratio (RER) atV˙o 2 max in trained mice. Exercise capacity and adaptations have also been measured indirectly, e.g., as maximal exercise duration and total distance run (26). A 4-wk regimen led to 15% increased cardiac mass as well as increased expression of hypertrophy markers, i.e., atrial and brain natriuretic peptides, by 74 and 52%, respectively (1). Myosin heavy chain redistribution toward greater expression of type IIa and IId/x occurred, whereas skeletal muscle weights remained unchanged. Four weeks of swimming led to cardiac hypertrophy, with a 10% increase in heart weight and a 16% increase in heart-to-body weight ratio (24). Although some studies have reported problems with both acute and chronic mice training, the large number of training studies in mice indicates the opposite. However, procedures for reliable exercise capacity measurements have been difficult to establish in mice (e.g., Refs. 11, 26).

In contrast, rat studies on training effects are numerous, with well-defined protocols for exercise capacity evaluation, intensity-controlled treadmill running, and robust cardiovascular outcomes. Until recently, findings appeared somewhat contradictory. Treadmill running with fixed exercise intensity induced only minor increments in ventricular mass and cardiomyocyte dimensions (2,3, 28, 29) or did not induce any effects (16, 18). However, 10–20% improvements inV˙o 2 max and work economy had been demonstrated (16, 30). Because swim training had produced up to 33% ventricular enlargement in rats, some studies concluded that the mode of exercise is crucial for eliciting cardiac training adaptations (16, 18). Even in rats, few studies had accounted for the fact that the load intensity required to induce physical conditioning increases as the performance improves during the course of training (4). However, a recent study from our laboratory demonstrated that intensity-controlled treadmill running induced some of the largest and most consistent effects of exercise training (42).

The purpose of the present study was to establish an experimental model of intensity-controlled treadmill running in mice. The specific aims were to 1) establish a valid and reliable treadmill protocol for evaluating endurance capacity in mice; 2) establish a standardized, long-term, intensity-controlled treadmill running protocol that is appropriately adjusted to the individual mouse endurance capacity by weekly V˙o 2 maxassessments; and 3) determine the effects of exercise training on endurance capacity and cardiac adaptations, as assessed by echocardiography and postmortem characteristics in male and female mice.


Study population and design.

A total of 40 female and 36 male adult C57BL/6J mice, 19–23 g, 7–8 wk old (Møllegaards Breeding Center, Lille Skensved, Denmark), were included in the study. Animals were maintained in a 12:12-h light-dark cycle, 22.5 ± 1.4°C temperature, and 55.6 ± 4.0% relative humidity. The mice were fed a pellet rodent diet and water ad libitum. Strewment was changed every third day. All experimental protocols were performed during the mice dark cycle, and the animals were rewarded with chocolate (Crispo, Nidar Bergene, Norway) after each training or test session. Sedentary mice were given the same amount of chocolate. None of the mice was excluded in the study because they avoided treadmill running, and the same person handled the mice during the study. The experimental protocol was approved by the Norwegian Council for Animal Research, and the experimental procedures conformed with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the Guiding Principles for Research Involving Animals and Human Beings from the American Physiological Society. The mice were assigned into groups as described in Table1.

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Table 1.

Group assignment and number of mice in each protocol

Aerobic capacity.

Oxygen uptake (V˙o 2) in mice was tested during treadmill running in a metabolic chamber. Ambient air was led through the chamber at a rate of 0.5 l/min, and 200 ml/min samples of gas were extracted to the paramagnetic oxygen analyzer (type 1155, Servomex) and the carbon dioxide analyzer (LAIR 12, M & C Instruments). The gas analyzers have an accuracy of measurements of ±2% and were calibrated with standardized gas mixtures before every test session. The single-lane test treadmill was custom made and placed in the metabolic chamber; for training, mills with multiple lanes were used. Stainless steel grids at the end of the lines provided an electrical stimulus of 0.25 mA, 1 Hz, and 200-ms length, to keep the mice running, and brushes avoided the mice from pinching feet between grid and treadmill. Test-retest assessments of V˙o 2and RER were performed during submaximal running at fixed standardized treadmill velocities at 25° (47%) inclination of the treadmill. After having been exposed to the equipment and treadmill running twice, six mice of both sexes were assigned to test-retest procedures. Mice were placed in the metabolic chamber for 10 min, and then they went through a 10-min warm-up at 0.08 m/s before running for 5 min at four different velocities (0.1, 0.14, 0.18, and 0.22 m/s). Each mouse ran twice, with the velocity order low to high or high to low set randomly, and performed both. With modifications, this method was adopted from previous studies in rats (6, 42).

To establish an optimal test protocol for measuringV˙o 2 max, six untrained female and male mice were tested at five different inclinations (0, 15, 25, 35, and 45°), one test each day and the inclinations in random order, to avoid possible conditioning biases. Each mouse had a regular 20-min warm-up at 40–50% of V˙o 2 maxbefore the V˙o 2 max protocol, which was carried out by increasing speed by 0.03 m/s every 2 min. Because the tests showed that middle inclinations (15–35°) resulted in highest peak V˙o 2, these inclinations (15, 20, 25, 30, and 35°) were tested again after 8 wk of training in six female and male mice, one test each day and the inclinations in random order, to determine whether V˙o 2 maxoccurred at similar grades.

o 2 max was measured at the start of every training week, and before and after the experimental training period, whereas running economy was measured before and after. BeforeV˙o 2 max testing, all mice went through a regular warm-up before they ran at fixed submaximal velocities of 0.15, 0.20, and 0.25 m/s at 25° inclination, for 5 min at each level, to determine the running economy. The treadmill velocity was then increased by 0.03 m/s every second minute until the mice were unable to, or refused to, run further. The criterion for reachingV˙o 2 max was whenV˙o 2 leveled off despite increasing running velocity. Another criterion for reachingV˙o 2 max was RER above 1.0.


For the investigations of prolonged training adaptations, 28 female and 24 male mice were randomized into either treadmill running or sedentary control groups. When V˙o 2 max remained unchanged for at least 3 consecutive wk, which occurred after 8 wk of training, the mice were killed. Training mice exercised on treadmills 2 h/day for 5 days/wk. At the start of every week,V˙o 2 max was measured as described and workloads adjusted accordingly. In training mice, exercise intervals alternated between 8 min at 85–90% ofV˙o 2 max and 2 min at 50–60%. Before the first interval, each mouse performed a regular warm-up as described earlier. At the day whenV˙o 2 max was tested, trained mice performed eight intervals after the test. In sedentary mice, treadmill running skills were maintained by treadmill running for 15 min on a flat treadmill at 0.15 m/s for 3 days/wk. This latter activity did not seem to alter either V˙o 2 max or work economy (Fig. 3).


Echocardiography was performed in 16 female mice, after sedation with ketamine hydrochloride (100 mg/kg, Pfizer) and xylazine (5 mg/kg, Bayer) intraperitoneally, by using a GE Vingmed System FiVe ultrasound scanner with an epicardial probe at 10 MHz and 250 frames/s (GE Vingmed Ultrasound, Norway). After the mouse chest was shaved, the transducer was gently placed on it for recordings. Heart rate, systolic and diastolic left ventricular wall thickness, lumen diameters, and fractional shortening were calculated as the mean of 10 consecutive cardiac cycles in two-dimensional M-mode long-axis recordings following the recommendations of the American Society of Echocardiography (35). Mitral inflow deceleration time, peak velocity of early and late component of mitral inflow, and isovolumetric relaxation time were calculated as the mean of 10 consecutive cardiac cycles of pulsed wave Doppler spectra recordings.

Cardiac and skeletal muscle mass.

After the training, mice were anestethized with diethyl ether and anticoagulated (0.1 ml heparin, 1,000 IU/ml; Novo Nordisk, Cophenhagen, Denmark). Hearts were rapidly removed, and left and right ventricles were carefully dissected and weighed. Extensor digitorum longus and soleus muscles from both hindlimbs were dissected out carefully and weighed after tendon removal.

Left ventricular myocytes.

After the training, mice were anestethized and heparinized, and hearts were removed as described above. Hearts were then submerged in oxygenated perfusion buffer, cannulated via the aorta under a microscope, and connected to a standard Langendorff retrograde perfusion system adapted to mice. To balance variation of the left ventricle myocyte isolation method, one heart from both trained and sedentary group was taken each day. Left ventricular and septal myocytes were isolated by using a modified protocol from Christensen et al. (9). The heart was perfused at 2.2 ml/min for 10 min with a modified Joklik's minimum essential medium (Life Technologies) mixed in 2,000 ml deionized water containing (in mM) 1.2 MgSO4, 1.0 dl-carnitine (Sigma Chemical), and 23.8 NaHCO3. The buffer was equilibrated with 5% CO2-95% O2 for 30 min (37°C, pH 7.2). Subsequently, 150 U/ml collagenase type II (Worthington) and 0.1% (wt/vol) bovine serum albumin (Sigma Chemical) were added, and the heart was perfused until it became palpably flaccid, which usually occurred after 6–8 min. The heart was then cut down into the Joklik's medium containing 0.8% (wt/vol) bovine serum albumin (Sigma Chemical) and 0.75 mM CaCl2. The left ventricle was separated and minced and was shaken for 15–20 min in the collagenase type II and bovine serum albumin buffer (37°C, 5% CO2-95% O2, 100 rpm). The solution with the myocytes and unsolved tissue was then gently filtered through a nylon mesh (250 μm); added to HEPES buffer containing (in mM) 135 NaCl, 5 KCl, 1 MgCl2 · 6H2O, 1.2 CaCl2 · 2H2O, 10 HEPES, 8 C6H12 · H2O (37°C, pH 7.2, 5% CO2-95% O2); and centrifuged (600 rpm, 45 s, 21°C). The supernatant was then removed, and HEPES buffer was added. Cardiomyocytes were deposited on laminin-coated coverslips (10 mg/ml, Life Technologies) and placed in a cell chamber on an inverted microscope (Diaphot-TMD, Nikon) and measured for length and midpoint width.

Allometric scaling.

As demonstrated in Table 3, the body mass increased more in males than in females. Changes in ventricular and skeletal muscle weights and V˙o 2 may not be entirely due to training regimens but also may be due to growth-related changes in body mass. It was therefore necessary to normalizeV˙o 2 and cardiac and skeletal muscle weights to the body dimensions (4). According to dimensional analysis and empirical studies,V˙o 2 should be expressed in relation to body mass raised to the power of 0.75, over a wide range of body weights (e.g., Ref. 41). Dividing for instance organ weight by body weight is the usual approach, but this has previously been shown to be valid only if body weight is expressed as lean body mass (5), because fat has very low metabolic activity (4). When lean body mass is undefined, ventricular mass should be expressed in relation to body mass raised to the power of 0.78, which is demonstrated empirically as the best approximation (5). Because no empirical studies relate the hindlimb muscles of the mice to body weight, the reduced exponent was calculated in the present training and sedentary control population by allometric equations to determine the relationship between body and extensor digitorum longus and soleus muscle mass. Skeletal muscle mass =a · mb , where a is the mass coefficient, m is the body mass, and bis the reduced exponent. The numerical value of bcan be obtained from the log-log plot of the experimental data because the logarithmic expression is a straight line (e.g., log soleus mass = log a + b · logm). It was found that skeletal muscle mass should be expressed to body mass raised to the power of 0.75. A detailed introduction to allometric scaling is presented elsewhere (4).


Data are expressed as means ± SD. The Friedman test and appropriate procedures for multiple comparisons were used to determine changes in V˙o 2, RER, and body weights throughout the experimental period, as well as differences inV˙o 2 by using different inclinations of the treadmill. The Mann-Whitney U-test was used to evaluate differences between groups and sexes. The relationship betweenV˙o 2 and speed was calculated by linear regression analysis. P < 0.05 was considered statistically significant.


Test protocols.

The procedures for measuring V˙o 2 and RER were found to be reproducible for measuringV˙o 2 and RER during exercise. Measurements of V˙o 2 in both female and male mice running at four submaximal velocities were similar on 2 different days (Fig. 1). Test-retest correlationV˙o 2 was 0.98, and the coefficient of variation was 9.1%. Inclination of the treadmill affected the highestV˙o 2 measured, as demonstrated in Table2. This was evident in both trained and untrained mice of both sexes. Peak V˙o 2was found at a range of inclinations between 15 and 35°. RER reached the highest values in the same range of inclinations, although in untrained females the maximum value occurred at 25° (47%) inclination (Table 2). Testing procedures forV˙o 2 max were reliable and consistent with previous results. As shown in Fig.2, V˙o 2increased linearly with increasing running velocity, and reached a plateau despite increased velocity, in both trained and untrained mice of both sexes. Linear regression analysis was performed untilV˙o 2 leveled off for both sexes [i.e., for trained female mice, V˙o 2 = 110.3 · speed + 13.6 (r = 0.98,P < 0.001), and for sedentary controls,V˙o 2 = 49.2 · speed + 33.1 (r = 0.97, P < 0.001)]. The respective linear regression for trained male mice wasV˙o 2 = 51.9 · speed + 36.7 (r = 0.99, P < 0.001), and for their respective sedentary controls,V˙o 2 = 29.3 · speed + 45.2 (r = 0.96, P < 0.001).

Fig. 1.

Test-retest of oxygen uptake (V˙o 2) during treadmill running at submaximal intensities (0.10, 0.14, 0.18, and 0.22 m/s) in female (n = 6) and male (n = 6) mice. No differences were found between tests or sexes. CV, coefficient of variation. Individual data are shown.

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Table 2.

Peak oxygen uptake and respiratory exchange ratio at different treadmill inclinations

Fig. 2.

o 2 at increasing intensity during treadmill running at 25° inclination in female (A) and male (B) trained (Tr; n = 8 female and 8 male) and sedentary (Sed; n = 8 female and 8 male) mice. Running was performed first in 5-min bouts at 0.15, 0.20, and 0.25 m/s and then with the speed increased from 0.25 m/s with 0.03 m/s every 3 min until exhaustion. Note thatV˙o 2 levels off before exhaustion, despite increasing intensity. Values are means ± SD.

Training effects.

After 6 wk of training, both female and male mice reached a plateau 49 ± 7 and 29 ± 5% above sedentary controls in female and male mice, respectively. However,V˙o 2 max appeared to increase progressively in both sexes when it was not corrected for body mass. As shown in Fig. 3, female mice had a higherV˙o 2 max than males, except in baseline recordings and in sedentary controls. This was evident both when expressed as milliliters per kilogram per minute and expressed as milliliters per kilogram raised to the power of 0.75 per minute but not when expressed as milliliters per minute. Body mass increased in all mice during the training regimen by 14 ± 3 and 17 ± 3% in trained and sedentary female mice, respectively, and by 29 ± 5 and 34 ± 6% in male counterparts (allP < 0.01; Table 3). Body mass did not develop differently between sedentary and trained mice within any sex, but increased significantly more in male mice than in females. When properly scaled for body mass,V˙o 2 max (expressed ml · kg−0.75 · min−1) in female mice was 15% higher than in males mice after the training regimen. The maximal running speed whenV˙o 2 max was tested increased from 0.36 ± 0.03 to 0.63 ± 0.03 m/s in female mice during the training regimen, whereas the respective increase in male mice was from 0.37 ± 0.02 to 0.65 ± 0.04 m/s.

Fig. 3.

Time course of maximal V˙o 2during training period in Tr (n = 14 female and 12 male) and Sed (n = 14 female and 12 male) mice.A: maximal V˙o 2 without normalization to body mass. B: maximalV˙o 2 normalized to body mass.C: maximal V˙o 2 normalized to body mass raised to the power of 0.75 according to allometric scaling. Tr mice ran 2 h/day for 5 days/wk in intervals of 8 min at 85–90% and 2 min at 50–60% of maximal V˙o 2, whereas Sed mice ran 15 min at 0° inclination at 0.15 m/s for 3 days/wk. Tr mice were tested at the beginning of every week, whereas Sed mice only performed pre- and posttraining tests. Note that maximalV˙o 2 levels off after 6 wk. Values are means ± SD. a Difference between trained and sedentary mice within the same sex, P < 0.01. Difference between sexes: b P < 0.01,c P < 0.05.

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Table 3.

Postmortem data of trained and sedentary mice

Work economy, i.e., V˙o 2 at a given running intensity, improved by 24.7 ± 3.4% in females and by 18.7 ± 4.6% in males after the training period (Fig.4 A). Training also reduced RER during submaximal running by 8.7 ± 1.1% in females and 8.5 ± 1.0% in males. The decrease in RER was most marked at the highest intensities (Fig. 4 B). There was a trend (P= 0.14) toward a lower oxygen cost and RER in female mice during identical submaximal running velocities (Fig. 4).

Fig. 4.

Work economy in Tr (n = 14 female and 12 male) and Sed (n = 14 female and 12 male) mice after 8 wk, i.e., V˙o 2 (A), and respiratory exchange ratio (B) during treadmill running at 25° inclination and 0.15, 0.20, and 0.25 m/s. Tr mice ran 2 h/day for 5 days/wk in intervals of 8 min at 85–90% and 2 min at 50–60% of maximal V˙o 2, whereas Sed mice ran 15 min at 0° inclination at 0.15 m/s for 3 days/wk.V˙co 2, carbon dioxide production. Values are means ± SD. Note that Tr mice differed significantly from Sed in either sex. a Difference between trained and sedentary mice within the same sex, P < 0.01.

In trained females, right and left ventricular mass increased by 28.9 ± 2.0 and 19.1 ± 1.2%, respectively, whereas the corresponding increments in males were 17.2 ± 1.4 and 12.3 ± 0.9% (Tables 3 and 4). Myocardial hypertrophy was confirmed in cardiomyocytes isolated from left ventricles in both sexes (Table 3). Cell length and width increased by 20.5 ± 2.3 and 32.2 ± 2.8% in females, and 17.1 ± 1.9 and 23.0 ± 3.1% in males, respectively. Cardiac weights were greater in males than in females, whereas there were no sex differences in cardiomyocyte dimensions. When echocardiographic measurements were performed in female mice, a 16.6 ± 2.2% increase in systolic left ventricular posterior wall thickness was detected (Table5). Systolic intraventricular septum thickness and diastolic left ventricle diameter showed trends toward increase (9.0 ± 1.6%, P = 0.17 and 8.2 ± 1.8%,P = 0.17, respectively).

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Table 4.

Ventricular and skeletal muscle weights scaled to body mass

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Table 5.

Echocardiographic measurements in anesthetized female mice

Skeletal muscle mass increased in trained mice of both sexes (Tables 3and 4). The weights of the hindlimb extensor digitorum longus and soleus muscles increased by 12.0 ± 1.1 and 15.5 ± 2.6% in females, and 16.4 ± 3.0 and 18.2 ± 2.7% in males, respectively. The mass of the extensor digitorum longus muscle was significantly greater in males, also when scaled appropriately (Tables3 and 4). Log-log plots for determining the relationship between the extensor digitorum longus muscle and the soleus muscle showed the reduced exponent to be 0.75 ± 0.06 (r = 0.56,P < 0.01) for the extensor digitorum longus and soleus muscles.


The present study documents robust and well-controlled protocols for exercise training of mice of both sexes. ReliableV˙o 2 max assessments were obtained in treadmill running at 25° (47%) inclination, whereas peakV˙o 2 measurements at grades below 15° or above 35° underestimated aerobic exercise capacity. Within 6 wk, intensity-controlled interval running at this inclination induced the largest increments in V˙o 2 max, myocardial weights, and cardiomyocyte dimensions reported in this species. As discussed in Aerobic capacity after training, allometric scaling significantly facilitated comparison across sexes and varying body weights. We found that noninvasive evaluation of cardiac dimensions by echocardiography was less sensitive in detecting myocardial hypertrophy than postmortem chamber weights and assessments of isolated cardiomyocyte length and width by video microscopy.

Test protocols.

The most common method for testing aerobic capacity in mice is recording volume and velocity of voluntary wheel running (15, 17,19, 34, 44). Others have estimated aerobic capacity by total time of standardized incremental treadmill running (e.g., Ref.26), V˙o 2 during swimming (25), or by the cold-exposure method (e.g., Refs.20, 25, 38). However, these approaches have several problems. Seeherman et al. (38) found that the cold-exposure method yielded 23% lowerV˙o 2 max than measurements during treadmill running, whereas swimming differs from running because of relatively inactive forelegs (24). Aerobic capacity may further be underestimated if motivation is low.

Test-retests of V˙o 2 and RER demonstrated reliable protocols. Similar values were obtained on separate days during standardized running, which is in accordance with previous studies in rats in our laboratory (42). A 5-min stage at each intensity was chosen, because V˙o 2leveled off after ∼3 min (data not shown), as previously demonstrated in rats (42) and humans (4).V˙o 2 and RER were therefore recorded during the last 2 min of each stage.

As reported in rats (42), we found an inclination-dependent peak V˙o 2 between 15 and 35° in both trained and sedentary mice. In mice, angle seems to be less crucial than in rats and humans, but RER indicated that 25° inclination yielded the highest cardiovascular load. A high load is important because inappropriate treadmill inclination might underestimate training-induced adaptations if a trueV˙o 2 max is not reached. A graded treadmill recruits a larger muscle mass, lowers the cadence, and induces RER values above 1.0. We suggest that flattening off ofV˙o 2 and RER above 1.0 may be used as criteria for V˙o 2 max in mice. As also was reported previously in rats (6, 42),V˙o 2 increased linearly with running velocity. This linearity thus provides a tool to estimateV˙o 2 from running velocity. However, the linear regression should only be used if identical inclinations, strains, and training states are compared.

o 2 max in untrained mice has been reported to range from ∼80 to 260 ml · kg−1 · min−1, and RER atV˙o 2 max from 0.91 to 1.28 (13, 14,31, 32, 36, 38, 40). However, there are differences in these studies regarding test protocols, equipment, body masses, strains, age, sex, and how accustomed the mice were to the methods. For instance, it has been shown that mice strain (e.g., Refs. 12,26), ambient temperature (e.g., Ref. 32) and age (e.g., Ref. 36) affectV˙o 2 max. On the other hand, the two studies investigating V˙o 2 max and RER in C57BL/6J, age and test protocol as the present study, found similar results (12, 27).

Aerobic capacity after training.

The training regimen in the present study is probably the largest and most efficient reported. Within 6 wk, V˙o 2reached a plateau 50% above sedentary controls in female mice and 30% above controls in males. Niebauer et al. (31) found thatV˙o 2 max increased 10% in C57BL/6J females after treadmill running for 4 wk, 1 h twice each day, 6 days/wk, at an intensity of ∼85% ofV˙o 2 max. Increments of 5–6% in C57BL/6J males (37) and 12% occurred in male Hsd:ICR house mice (40) after 6 wk of treadmill running and 7–8 wk of voluntary wheel running, respectively. Differences in training adaptations reported in other studies are probably due to different training regimens and protocols and to insufficient control of exercise intensity. However, comparison of the results in many previous studies may be confounded by different methods for normalizingV˙o 2 to body weight. As shown in Fig. 3, allometric scaling greatly enhances comparison ofV˙o 2 in animals with different weights. Reports of long-term running in mice have reported decreased (34,40, 44) or unchanged (1, 19, 31, 33, 37, 39) body mass, whereas swimming yielded an increase (24). In a survey of 100 recent transgenic mice studies at PubMed, body mass ranged between 17 and 74 g and age between 26 days and 12 mo.

In the present study, work economy and RER increased in line with rats and humans (4, 30, 42). Work economy and RER have not been measured in trained mice previously, because most investigations of long-term training effects only report increased velocity and distance run (e.g., Refs. 1, 23, 41).

Whereas V˙o 2 max is regarded as the best denotation of aerobic capacity (4), work economy provides a tool to investigate other aspects of aerobic capacity and chronic conditioning. Endurance training at lower intensities may substantially improve work economy, without increasingV˙o 2 max. Improved work economy probably contributes to the large increase in maximal running velocity atV˙o 2 max.

Cardiac structure and function.

In the present study, ventricular weights increased more than previously reported in mice and about the same as in rats exercising for 7 wk with the same regimen (42). Other studies in mice report no change (31) or an increase between 10 and 15% in cardiac mass (1, 24), after 4 wk of training in C57BL/6J mice, treadmill, voluntary wheel, and swimming exercise, respectively. These studies lack a rigorous control of exercise intensity, and cardiovascular load seemed lower.

As far as we know, this is the first report of training-induced cardiomyocyte growth in mice. Both cell length and width increased, whereas only lengthening was observed in rats (29, 42). No differences in cardiomyocyte dimension between the sexes were found, even though males had greater ventricle mass, which may suggest a higher number of cardiomyocytes in males.

Noninvasive assessment of hypertrophy by echocardiography was less sensitive than postmortem measurements. A modest increase in systolic left ventricular posterior wall thickness, and trends in systolic intraventricular septum thickness and diastolic left ventricle diameter were detected. Ultrasound M-mode or Doppler measurements detected no other changes. The lower sensitivity in echocardiography may result from insufficient temporal or spatial resolution. Echocardiography with a 15-MHz linear probe indicated that 49 animals would be required to detect 15% (power ≥ 0.8, α of 0.05) differences in M mode between two groups of CD-1 mice, hypertrophy and control (10). Compensatory mechanisms in vivo at rest and negative inotropic and chronotropic effects of anesthesia are other possible causes. Echocardiography revealed bradycardia, as previously observed in mice (7, 43).

Skeletal muscle.

The weights of the hindlimb extensor digitorum longus and soleus muscles increased substantially. Males had larger extensor digitorum longus muscles, but soleus muscle weight was similar. Other studies report varying results: 20–24% increased (39) and unchanged (1, 19) skeletal muscle mass, and increased cross-sectional area (19, 33) and GATA-2 expression (hypertrophy marker), after 4–12 wk of training (19,33). Interestingly, skeletal muscle growth occurs in mice in response to endurance training, contrary to observations in humans (4).


In summary, the present study documents a procedure for reliable testing of V˙o 2 max in mice. Graded treadmill running seems to be a sensitive method to assess changes in work capacity and work economy in response to exercise training and probably also in disease states such as heart failure. The protocol for intensity-controlled interval treadmill running provides a well-defined model for exercise training that yields large and robust effects that mimic central results in humans. We anticipate that future experiments in this model will unveil novel molecular, cellular, and integrative mechanisms of adaptation to exercise, e.g., differences in signaling pathways between training-induced adaptive myocyte enlargement and pathological hypertrophy observed in heart failure. Understanding the mechanisms of training-induced amelioration of myocardial function may help identify molecular targets for the treatment and prevention of cardiovascular disease.


The authors thank Arnfinn Sira and Ketil Jensen for building the mice testing and training equipment, Bjørn Angelsen and Hans Torp for help with echocardiography, and Jan Helgerud for advice on allometric scaling.


  • This work was supported by grants from the Norwegian Council on Cardiovascular Diseases, the EWS Foundation, the Torstein Erbo Foundation, and the Arild and Emilie Bachkes Foundation. O. J. Kemi is the recipient of a research fellowship from the Norwegian University of Science and Technology.

  • Address for reprint requests and other correspondence: Ø. Ellingsen, Dept. of Physiology and Biomedical Engineering, Medical Technology Center, Norwegian Univ. of Science and Technology, Olav Kyrres gate 3, N-7489 Trondheim, Norway (E-mail:oyvind.ellingsen{at}

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • May 24, 2002;10.1152/japplphysiol.00231.2002


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