Subjects
Eleven untrained subjects (7 women and 4 men) with an average age (ranged 36–53 years), body mass, and VO2 max of 42.7 ± 1.5 year, 78.4 ± 4.4 kg, and 38.3 ± 1.6 ml/kg/min, respectively, participated in the study. To be included in the study, the adults should be nonsmokers, not having done regular training for the last 2 years and performing less than 2 h of physical activity per week, and not having a VO2 max above 40 and 45 ml/min/kg for women and men, respectively. During the intervention period, the subjects did not change their daily activities but participated in the 5–10–15 training. The physical characteristics and VO2 max of the subjects who completed the intervention are presented in Table 1. During the INT, one woman left the study due to injury, a woman withdrew due to disease in the family, and another woman was not included in the data due to injury and, therefore, low compliance to training.
Table 1 Characteristics of subjects
All participants were fully informed of experimental procedures and any discomforts associated with participating in the study before signing a written informed consent. The study was approved by the Ethics Committee of Copenhagen and Frederiksberg communities and conformed to the code of the Ethics of the World Medical Association (Declarations of Helsinki) and the Title 45, U.S. Code of Federal Regulations, Part 46, Protection of Human Subjects, Revised November 13, 2001 (Revised June 23, 2005, effective June 23, 2005).
Experimental design
In a 7-week intervention period (INT), the subjects conducted 5–10–15 training (see Training) three times per week. Three weeks prior to the start of the study start subjects, underwent the following familiarization tests to avoid a learning effect when repeating the test after the INT: (1) a treadmill test to determine VO2 max; the velocity which elicited VO2 max (maximal aerobic speed, MAS); and time to exhaustion, (2) a 1500-m run, (3) the Yo–Yo Intermittent Endurance test level 1 (Yo–Yo IE1). Before the start and immediately after the end of INT, the aforementioned tests were replicated and a 3-km run was added to the test battery. In addition, on a separate day before and after the INT, subjects reported to the laboratory after an overnight fast to a dual-energy X-ray absorptiometry (DEXA) scan and a fasting blood sample.
Training
Training was conducted during spring and early summer in a park area on a smooth path of pebble and gravel. Every training session consisted of a standardized 15-min warm-up, including pair exercises and running drills (heel kicks, knee lifts, and side hops) followed by two 100-m runs with acceleration. After the warm-up, subjects did repeated 2-min periods of 5–10–15 running interspersed by 1 min of rest. In the first week, subjects performed three 2-min periods. The total number of 2-min periods increased by 1 every week until subjects completed nine running periods in the final week of the INT. Each 2-min period of 5–10–15 running consisted of 4 consecutive 30-s intervals divided into 15, 10, and 5 s at a running speed corresponding to ~ 30, ~ 50, and ~ 85% of highest maximal speed during training as determined with 5-Hz GPS. In between the 2-min periods, the subjects had a passive recovery for 1 min. The GPS units were placed into the manufacturer-designed harness, which was worn during training. After GPS recording during training, the data were downloaded and analyzed using proprietary software (Sprint, Catapult Sports, Canberra, Australia). Subjects ran for 29 and 15% of the running time with a speed above 85 and 100%, respectively, of the velocity that elicited maximal aerobic speed, respectively. The subjects completed 2.3 ± 0.12 training session per week during INT. The training distance was measured with a 5-Hz GPS system at a minimum of two occasions for each subject within the first 2 weeks of training and the last 2 weeks of training. The average volume of the 5–10–15 running of each subject increased from 2160 ± 115 m/week in the first week to 6479 ± 345 m/week in the final week (warm-up not included) of INT. Subjects were wearing heart rate monitors at least at two training sessions during INT. Heart rate response was related to the calculated maximal heart rate (220—age beats per min) or individual peak heart rate obtained during training.
Performance measurements
Before and within the first 14 days following the 7-week intervention period, the subjects completed testing with no more than 4 days between the tests. Prior to testing, the subjects refrained from severe physical activity for at least 48 h. The warm-up before conducting the Yo–Yo IE1, 1500-m, and 3-km run consisted of 15 min with low-to-moderate speed running including running drills (heel kicks, knee lifts, and side hops), dynamic stretches and mobilizing exercises. Before each test, the subjects was told to complete the 1500-m and 3-km test as fast as possible and continue as far as possible in the Yo–Yo IE1 test. The subjects had no information about their individual performance before completion of the last test in the study.
1500-m and 3-km
The 1500-m and 3-km run was carried out on a GPS measured 1.5-km course. The subjects did not wear watches during the tests and were not aware about running time. The time to complete the 1500 m and 3 km was used as the test result.
Yo–Yo intermittent endurance test level 1
On a separate occasion, the subjects completed the Yo–Yo IE1 test consisting of repeated 20-m shuttle runs, back and forth between the starting line and finish line marked by cones, at a progressively increased running speed dictated by audio bleeps from a pre-recorded source. Between each shuttle, the subjects had a 5-s rest period of slow jogging or walking around a cone placed 2.5 m from the starting line. The test result was recorded as the distance covered at the point when subjects have failed twice to reach the finishing line in between the bleep. All testing sessions were performed indoor on 2 × 20-m running lanes marked by cones (Bangsbo 1994).
Submaximal and incremental test
A familiarization test was carried out as an incremental test to exhaustion. The subjects started at a velocity of 5–8 km/h based on individual exercise history. The speed was gradually increased until volitional exhaustion. MAS was determined as the speed, which elicited VO2 max during the familiarization test. The test was performed on a motorized treadmill under standard laboratory conditions. On a second occasion, the subjects completed a submaximal treadmill running test followed by an incremental test to exhaustion. The subjects arrived at the laboratory, and before start of the test, the subjects had a catheter (18 gauge, 32 mm) inserted in an antecubital vein. The test consisted of two 5-min runs corresponding to 70% of MAS (8.0 ± 0.6 km/h) and 85% of MAS (10.1 ± 0.5 km/h), separated by a 2-min rest period. Submaximal running speeds were estimated from the determination of MAS obtained during the familiarization test. Two subjects walked at 5 km/h, instead of running, as 70% of MAS for these subjects was below 7 km/h. Two minutes after the cessation of the second submaximal bout, subjects carried out an incremental test starting with 1 min at a speed of 85% of maximal aerobic speed. Hereafter, running speed increased by 0.5 km/h every minute until volitional exhaustion. The time to exhaustion was measured from the beginning of the incremental test and until volitional exhaustion, and the time used as the test result. Pulmonary VO2 was measured throughout the exercise periods by a breath-by-breath gas analyzing system (Oxycon Pro, Viasys Healthcare, Hoechberg, Germany), which was calibrated before each test. VO2 max was determined as the highest value achieved over a 30-s period. To meet the criteria for achievement of VO2 max at least two of the following three criteria should be achieved: (1) plateau in VO2 despite a further increase in speed; (2) a respiratory exchange ratio (RER) > 1.10 and (3) lactate concentration above 10 mmol/l after completing the incremental test to exhaustion. To determine blood lactate concentration during the submaximal and incremental test, blood samples were collected before the first bout, 1 min after each submaximal running bout as well as 30, 90, and 180 s after exhaustion using 2-ml heparinized syringes. Blood was analyzed for lactate concentration using an ABL 800 Flex (Radiometer, Copenhagen, Denmark) immediately after testing.
DEXA scan and resting blood samples
Subjects reported to the laboratory between 6 and 9 a.m. on a separate day after an overnight fast. Body composition was assessed from whole-body DEXA scanning (Prodigy Bone Densitometer System; GE Lunar, Madison, WI) with measurements of lean body mass, total fat mass, regional fat mass, bone mineral content (BMC), and bone mineral density (BMD) according to standard procedures. It has previously been reported that the precision error of the DEXA scanner is between 0.6 and 1.8% (Shepherd et al. 2006). Fasting blood was taken from the antecubital vein and collected to determine the concentration of bone turnover markers. A total of 1.5 mL of blood collected from a non-heparinized syringe was diluted in 30 mL EDTA and centrifuged for 2 min, after which the plasma was pipetted and frozen at − 80 °C until analyzed for bone markers. Serum/plasma Carboxy-terminal collagen crosslinks (CTX) was measured using the IDS-iSYS CTX (CrossLaps®) assay (Immunodiagnostic Systems, plc, Tyne and Wear, UK). Serum/plasma procollagen-type I N propeptide (P1NP) was measured using the IDS-iSYS intact P1NP assay (Immunodiagnostic Systems). Finally, serum/plasma osteocalcin was measured using the N-Mid Osteocalcin assay (Immuodiagnostic Systems). All assays were carried out on a dedicated automated analyzer, iSYS (Immunodiagnostic Systems) according to the manufacturer’s instructions. All three assays are chemiluminescence immunoassays.
All analysis were done with serum/plasma as the sample material. For each assay, the sample aliquots were kept frozen at − 80 °C until the day of analysis. None of the samples had previously been thawed, and all analysis were performed immediately after thawing the samples. All samples were analyzed using one single batch of each assay. Assay performance was verified using the manufacturers’ control specimens. The intermediary precisions expressed as coefficients of variation for CTX were 5.3% (at CTX concentration 213 ng/L), 3.4% (869 ng/L), and 3.5% (2113 ng/L) for iSYS. For P1NP, the intermediary precisions were 5.4% (18.96 µg/L), 6.5% (48.48 µg/L), and 6.1% (122.10 µg/L) for iSYS. Finally, for osteocalcin, the intermediary precisions were 3.0% (8.73 µg/L), 3.6% (27.6 µg/L), and 3.5% (68.7 µg/L). The reference values for Osteocalcin are 10.4–45.6 µg/L; P1NP are 27.7–127.6 µg/L. Furthermore, reference values for CTX are 0.115–0.748 µg/L for men and 0.112–0.738 µg/L in premenopausal women and 0.142–1.351 µg/L in post-menopausal women.
Statistics
Changes in performance (1500-m, 3-km, and Yo–Yo IE1), pulmonary VO2, fasting blood bone markers (Osteocalcin, CTX, and P1NP), and blood lactate were evaluated using a two-way analysis of variance for repeated measurement with a linear mixed model approach which was applied to the data using a the lme4 packages. Corresponding t tests and adjusted P values were calculated using the package multcomp. Measurements at the end of the intervention were used as dependent variables and measurements at baseline were the independent variables. A significance level of P < 0.05 was chosen. Data are presented as means ± standard error (SE). The R (R Core Team) statistic tool was used in all statistical analyses. To evaluate correlation (Pearson correlation) between training volume and changes in performance, pulmonary VO2, body composition, bone mineral density, blood bone turnover markers, and blood lactate, a Chi-squared distribution test was used.