DOI: 10.5061/dryad.31zcrjdv2
title: A comparison of new cardiovascular endurance test using the 2-minute marching test vs. 6-minute walk test in healthy volunteers: A crossover randomized controlled trial

description: This was a 2×2 randomized crossover control trial to compare the
 cardiovascular endurance of healthy volunteers using a 2-minute marching
 test (2MMT) and a 6-minute walk test (6MWT). This study included
 254 participants of both sexes, aged 20–50 years, with a height and body
 mass index (BMI) of ≥150 cm and ≤25 kg/m2, respectively. Participants
 could perform activities independently and had normal annual chest
 radiographs and electrocardiograms. A group-randomized design was used to
 assign participants to Sequence 1 (AB) or 2 (BA). The tests were conducted
 over 2 consecutive days, with a 1-day washout period. On day 1, the
 participants randomly underwent either a 6MWT or 2MMT in a
 single-anonymized setup, and on day 2, the tests were performed in reverse
 order. We analyzed maximal oxygen consumption (VO2max) as the primary
 outcome and heart rate (HR), respiratory rate (RR), blood pressure (BP),
 oxygen saturation, dyspnea, and leg fatigue as secondary
 outcomes. Data were collected from 127 participants, categorized
 into two groups for different testing sequences. The first (AB) and second
 groups had 63 and 64 participants, respectively. The estimated VO2max was
 equivalent between both groups. The 2MMT and 6MWT estimated VO2max with a
 mean of 41.00 ± 3.95 mL/kg/min and 40.65 ± 3.98 mL/kg/min, respectively.
 The mean difference was -0.35 mL/kg/min (95% confidence interval: -1.09 to
 0.38; p &lt;0.001), and no treatment and carryover effects were
 observed. No significant changes were observed in HR, RR, and systolic BP
 (p = 0.295, p = 0.361 and p = 0.389, respectively). However, significant
 changes were found in the ratings of perceived exertion (p &lt;0.001)
 and leg fatigue scale (p &lt;0.001). The 2MMT is practical,
 simple, and equivalent to the 6MWT in estimating VO2max.
description: <strong>Sample size</strong> The
 sample size required for the equivalence study was estimated using nQuery
 software and calculated using two one-sided equivalence tests for
 crossover design. To calculate the sample size, we set the alpha error
 probability, statistical power, the lower equivalence limit, and upper
 equivalence limit at 5%, 90%, -2.00, and +2.00, respectively, using the
 clinical margin (minimal clinically important difference [MCID] of
 VO<sub>2max</sub> from a previous study, which was 2 ml/kg/min
 [15], and standard deviation was 8.6 [16]. Based on these values, we
 needed 101 participants for the crossover design, allowing for a 20%
 dropout rate. Therefore, we decided to randomize 127 patients per arm,
 resulting in 254 participants. However, due to the COVID-19 pandemic, data
 collection was incomplete, and we could only analyze 127 data sets in this
 study. <strong>Inclusion and exclusion
 criteria</strong> The inclusion criteria were
 male and female healthy volunteers, aged 20–50 years, with height: ≥150 cm
 and, BMI ≤25 kg/m2. Participants could perform activities independently
 and had normal annual chest radiographs and electrocardiograms. The
 exclusion criteria were significantly unstable vital signs, a history of
 COVID-19, and underlying heart disease or neuromuscular/skeletal
 impairment. <strong>Procedure and measurement
 </strong> We conducted a 2MMT and compared the
 results with those of the standard test, the 6MWT, to test the equivalence
 of both tests in estimating VO<sub>2max</sub>
 <strong>Condition
 A</strong><strong>:</strong> According to the standard
 protocol, the 6MWT was performed indoors on a flat surface in a 30-m
 straight corridor, with 180º turns every 30 m. [10].<sup>
 </sup>The walk test was performed with stable vital signs, and
 SpO<sub>2</sub> was maintained at &gt;95%, all monitored
 by a cardiopulmonary physical therapist.
 <strong>Condition
 B</strong><strong>:</strong> The 2MMT was developed to
 determine the number of steps performed within 2 min. After the “start”
 command, the participants began marching in place and lifting their knees
 to an appropriate height of 30 cm. The participants were instructed to
 perform as many steps as possible (reaching a height of 30 cm) within 2
 min. The participants were allowed to perform a few training steps to
 adjust to the marching technique and verify their ability to complete the
 task. The participants marched at their own pace; they could slow down or
 even stop, if necessary, and continue marching until the end of the
 2-minute test period. The investigator determined the number of steps
 performed, informed the participants about the time left until the end of
 the trial, and motivated them to achieve the best possible result. The
 test results were expressed as the number of performed steps during which
 the right foot touched the ground. When the
 participants exhibited severe symptoms of exercise intolerance in both
 tests, such as severe dyspnea, fatigue, or other alarming symptoms, they
 were allowed to slow down or stop and rest. However, they were encouraged
 to resume the test as soon as possible. Adverse events were monitored
 during and after test completion. Both tests were terminated and
 interpreted as incomplete if any of the following symptoms were present:
 chest pain, intolerable dyspnea, leg cramps, staggering, diaphoresis, and
 ashen appearance. Data regarding the sex, age, BMI, HR,
 and RR were collected, and SpO<sub>2</sub> was assessed using
 the NONIN Onyx2 9590 Oximeter, SBP and DBP were measured using the Philip
 Patient Monitor Efficia CM100, RPE, and LFS were assessed using the Borg’s
 scale. All parameters were recorded at 1 min, 5 min, and 10 min for
 pretest and posttest. VO<sub>2max</sub>
 estimated the cardiovascular endurance using the following
 formula: VO<sub>2max</sub> estimated in the
 6MWT:<strong> </strong>70.161 + (0.023 × 6MWT [m]) - (0.276 ×
 weight [kg]) - (6.79 × sex, where m = 0, f = 1) - (0.193 × resting HR
 [beats per minute] - (0.191 × age [years]) [15]. where resting HR is the
 10-min resting HR of posttest.
 VO<sub>2max</sub> estimated in the
 2MMT:<strong> </strong>13.341 + 0.138 × total up and down
 steps (UDS) – (0.183 × BMI) [16]. <strong>Data
 analysis</strong> Due to the COVID-19 pandemic in
 Thailand and hospital policies, only 127 of the 254 participants, who were
 healthy volunteers, could complete data collection. Descriptive statistics
 were used to evaluate demographic characteristics. Continuous variables
 were reported as mean ± standard deviation, whereas binary variables were
 reported as percentages. The primary outcome
 (VO<sub>2max</sub>), evaluated using Statgraphics software,
 was analyzed through a two-one-sided t-test procedure. The analysis was
 conducted with an equivalence bound of ± 2 mL/kg/min from the margin of
 VO<sub>2max</sub> observed in a previous study [17]. The
 carryover and treatment effects were insignificant, and the equivalence
 result was significant for the test. For the secondary outcome, all
 parameters were analyzed using a linear mixed-effect model to compare 2MMT
 and 6MWT with STATA software. References
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description: # A comparison of new cardiovascular endurance test using the 2-minute
 marching test vs. 6-minute walk test in healthy volunteers: A crossover
 randomized controlled trial
 [https://doi.org/10.5061/dryad.31zcrjdv2](https://doi.org/10.5061/dryad.31zcrjdv2) We have submitted data tables 1-3 for the description of (Fig 1 CONSORT diagram of the study_figure.TIFF), (Fig 2 The trial design_figure.TIFF, and(Fig 3 The study protocol_figure.TIFF), and submitted of data analysis (Table 1 Baseline characteristics_data.CVS),(Table 2 Equivalence test of VO2max between 2MMT and 6MWT_data.CVS), (Table 3 Mean and standard deviation of 6MWT and 2MMT_data.CVS), and (Table 4 Comparison of secondary outcomes between 6MWT and 2MMT_data.CVS) ## Description ### **Fig 1 CONSORT diagram of the study**\Figure #### Materials and Methods The trial protocol and supporting Consolidated Standards of Reporting Trials (CONSORT) checklist are available as supporting information (S1 File CONSORT Checklist) and the CONSORT diagram is illustrated in Fig 1. ### **Fig 2 The trial design**\Figure #### Trial design We conducted a group of randomized controlled trials, including a crossover study for the equivalence test of cardiovascular endurance (VO2max) between the 6MWT and 2MM, with a 1-day washout period between the 6MWT and 2MMT. The trial design is outlined in Fig 2. ### **Fig 3 The study protocol**\Figure #### Overview of study design This study was conducted during two visits, 1 day apart. Notably, 254 healthy volunteers were enrolled. They were recruited through an announcement. All eligible participants were randomly assigned to one of two sequences using group randomization in a single anonymized setup. Sequences 1 (AB) and 2 (BA) involved the 6MWT and 2MMT, respectively. On day 1, the sequences were performed randomly, with a 1-day washout period between the tests. On day 2, the tests were performed in reverse order. The primary outcome, VO2max, was recorded in the posttest, and the secondary outcomes, including heart rate (HR), respiratory rate (RR), oxygen saturation (SpO2), systolic blood pressure (SBP), diastolic blood pressure (DBP), rating of perceived exertion (RPE), and leg fatigue scale (LFS), were recorded in 1 min, 5 min, and 10 min for pretest and posttest. The study protocol is outlined in Fig 3. ### **Table 1 Baseline characteristics**\_Data * *BMI*: body mass index * *SD*: standard deviation * *2MMT*:2-minute marching test * *6MWT*: 6-minute walk test ##### Characteristics: Notably,127 healthy volunteers completed the data collection and protocol without experiencing adverse events. The study involved 31 males and 96 females aged 20–49 years, with a mean age of 29.65 ± 5.85 years. The participants’ average weight, height, and BMI were 56.96 ± 8.02 kg, 163.92 ± 6.72 cm, and 21.05 ± 2.08 kg/m2, respectively. Participants had an average of 233.16 ± 46.94 steps for the 2MMT and an average distance of 565.48 ± 56.88 m. for 6MWT. The participants’ characteristics are presented in Table 1. ### **Table 2 Equivalence test of VO2max between 2MMT and 6MWT**\_Data * *2MMT*: 2-minute marching test * *6MWT*: 6-minute walk test * *VO2max*: maximal oxygen consumption * *SD*: standard deviation * *CI*: confidence interval * *_a*: Analyses were conducted using the two-one-sided t-test procedure (TOST). * *_b*: Analyses were conducted using a paired t-test. * \_*: Statistical significance set at p&lt; 0.05 Overall, 127 participants were included in this study; 64 participants underwent sequence 1 (AB) (6MWT followed by 2MMT), whereas 63 underwent sequence 2 (BA) (2MMT followed by 6MWT). The results are presented in Table 2. This study found that the 2MMT and 6MWT were equivalence in estimating VO2max The mean VO2max for the 2MMT was 41.00± 3.95 mL/kg/min, whereas it was 40.65 ±3.98 mL/kg/min for the 6MWT. The mean difference between the two tests was only 0.35 mL/kg/min, with a 95% confidence interval [CI] of -1.09 to 0.38 mL/kg/min. The p-value was &lt; 0.001, indicating no treatment or carryover effect. For sequence 1 (AB), the mean VO2max for both tests were 41.09 ± 4.13 mL/kg/min and 41.06 ± 3.88 mL/kg/min, respectively, with a mean difference of only 0.27 mL/kg/min (95% CI: 1.36, 1.42). The statistical analysis showed no significant difference between both methods (p-value 0.96), indicating that both tests yielded similar results for sequence 1 (AB). Similarly, for sequence 2 (BA), the mean VO2max was 40.87 ± 3.80 mL/kg/min and 41.29 ± 3.94 mL/kg/min for the 2MMT and 6MWT, respectively, with a mean difference of 0.58 mL/kg/min (95% CI: -0.50, 1.65). The statistical analysis was not significant (p-value = 0.28), indicating that both tests yielded similar results for sequence 2 (BA). ### **Table 3 Mean and standard deviation of 6MWT and 2MMT**\_Data * *2MMT*: 2-minute marching test * *6MWT*: 6-minute walk test * *HR*: heart rate * *RR*: respiratory rate * *SpO2*: oxygen saturation * *SBP*: systolic blood pressure * *DBP*: diastolic blood pressure * *LFS*: leg fatigue scale * *SD*: standard deviation ### **Table 4 Comparison of secondary outcomes between 6MWT and 2MMT**\_Data * *2MMT*: 2-minute marching test * *6MWT*: 6-minute walk test * *HR*: heart rate * *RR*: respiratory rate * *SpO2*: oxygen saturation * *SBP*: systolic blood pressure * *DBP*: diastolic blood pressure * *LFS*: leg fatigue scale * *CI*: confidence interval * *_a*: Analyses were conducted using a paired t-test * *_b*: Analyses were conducted using a linear mixed-effects model adjusted for baseline value * *_**: Statistical significance set at p&lt;0.05 Regarding the secondary outcomes, we compared the vital sign variables (HR, RR, SpO2, DBP, and SBP), dyspnea scale (RPE), and LFS which were analyzed using a linear mixed-effect model adjusted for baseline value. The results are outlined in Table 3 and Table 4. #### HR The comparison results in the posttest at 0 min of 2MMT and 6MWT presented a mean HR of 111.10 ± 21.82 bpm, and 105.81 ± 19.66 bpm, respectively. The 5 min posttest had a mean HR of 84.99 ± 12.75 bpm and 87.25 ± 11.72 bpm in 2MMT and 6MWT, respectively, and that for 10 min posttest was 82.73 ± 11.31 bpm and 84.64 ± 10.65 bpm, respectively. Changes in HR during the 2MMT had a statistically significant mean reduction of -2.672 (95%CI: -2.965, -2.379) (p-value &lt;0.001), and those in 6MWT had a mean reduction of -2.282. (95%CI: -2.575, -1.989), which was statistically significant (p-value &lt;0.001). The HR between the 2MMT and 6MWT had, a mean difference of -0.849 (95%CI: -2.438, 0.740) and was, not statistically significant (p-value = 0.295). #### RR The comparison results in the posttest at 0 min of the 2MMT and 6MWT presented a mean of RR 21.57 ± 2.64 bpm and 21.43 ± 2.58 bpm, respectively. The mean RR during the 5 min posttest was 18.48 ± 1.79 bpm and 18.22 ± 2.09 bpm in 2MMT and 6MWT, respectively, and that for the 10 min posttest was 17.35 ± 1.50 bpm and 17.19 ± 1.76 bpm, respectively. RR changes during the 2MMT had a statistically significant mean reduction of -0.415 (95%CI: -0.454, -0.376) (p-value &lt;0.001), and those in 6MWT had a mean decrease of -0.431 (95%CI: -0.469, -0.392) which was statistically significant (p-value &lt;0.001). The RR change between the 2MMT and 6MWT had, a mean difference of 0.121 (95%CI: -0.138, 0.379) and was, not statistically significant (p-value = 0.361). #### SpO2 The comparison results in the posttest at 0 min of the 2MMT and 6MWT presented a mean of SpO2 of 99.35 ± 0.97 % and 99.57 ± 0.72%, respectively. The mean SpO2 at 5 min posttest was 99.34 ± 0.90% and 99.44 ± 0.78% in 2MMT and 6MWT, respectively, and that for the 10 min posttest was 99.41 ± 0.84% and 99.31 ± 0.80%. Changes in SpO2 2MMT had a statistically non-significant mean reduction of -0.010 (95%CI: -0.024, 0.004) (p-value= 0.163), and those in the 6MWT had a non-significant mean decrease of -0.011 (95%CI: -0.024, 0.003) (p-value = 0.141) the change in SpO2 between the 2MMT and 6MWT, a mean difference of -0.106 (95%CI: -0.195, -0.017) was statistically significant (p-value = 0.020). #### SBP The comparison results in the posttest at 0 min of the 2MMT and 6MWT presented a mean SBP of 132.86 ± 16.87 mmHg and 135.06 ± 16.48 mmHg, respectively. The mean SBP at the 5 min posttest was 117.50 ± 12.65 mmHg and 117.20 ± 12.49 mmHg in 2MMT and 6MWT, respectively, and that for 10 min posttest was 114.95 ± 11.62 mmHg and 114.43 ± 10.48 mmHg, respectively. SBP changed during 2MMT had a statistically significant mean reduction of -1.838 (95%CI: -2.077, -1.600) (p-value &lt;0.001), and those during 6MWT had a mean decrease of -2.014 (95%CI: -2.253, -1.775), which was statistically significant (p-value &lt;0.001). The SBP changes between the 2MMT and 6MWT had, a mean difference of 0.560 (95%CI: -0.715, 1.834) and was, not statistically significant (p-value = 0.389). #### DBP The comparison results at 0 min of 2MMT and 6MWT presented a mean DBP of 70.85 ± 10.26 mmHg and 75.27 ± 11.17 mmHg, respectively. The mean DBP at the 5 min posttest was 70.57 ± 9.03 mmHg and 71.39 ± 9.15 mmHg in 2MMT and 6MWT, respectively, and that for the 10 min posttest was 70.28 ± 8.50 mmHg and 71.36 ± 9.57 mmHg, respectively. DBP changes during 2MMT had a statistically significant mean reduction of 0.263 (95%CI: -0.412, -0.114) (p-value = 0.001). The DBP changes in 6MWT had a mean reduction of 0.185 (95%CI: -0.334, -0.036), which was statistically significant (p-value = 0.015). The DBP changes between the 2MMT and 6MWT had a statistically significant mean difference of -1.759 (95%CI: -2.589, -0.928) (p-value &lt; 0.001). #### Dyspnea The dyspnea scale was presented using RPE and the comparison result in the posttest at 0 min of 2MMT and 6MWT presented a mean RPE of 1.96 ± 1.25 and 2.35 ± 1.40, respectively. The mean RPE at the 5 min posttest was 0.29 ± 0.51 and 0.56 ± 0.74 in 2MMT and 6MWT, respectively, and that for the 10 min posttest was 0.04 ± 0.16 and 0.11 ± 0.34 in 2MMT and 6MWT, respectively. RPE changes during 2MMT had a statistically significant mean reduction of 0.215 (95%CI: -0.237, -0.194) (p-value &lt;0.001), those during 6MWT had a statistically significant mean decrease of -0.201 (95%CI: -0.222, -0.180) (p-value &lt; 0.001), and those between the 2MMT and 6MWT had, a statistically significant mean difference of -0.227 (95%CI: -0.350, -0.105) (p-value &lt; 0.001). #### Leg Fatigue Scale LFS was modified from Borg’s scale, and the comparison results in the posttest at 0 min of 2MMT and 6MWT presented a mean LFS of 1.85 ± 1.23 and 2.11 ± 1.44, respectively. The 5 min posttest presented a mean of 0.42 ± 0.63 and 0.71 ± 0.83 in 2MMT and 6MWT, respectively, while the 10 min posttest had 0.11 ± 0.31 and 0.22 ± 0.53 in 2MMT and 6MWT, respectively. LFS changes during 2MMT had a statistically significant mean decrease of 0.193 (95%CI: -0.216, -0.169) (p-value &lt;0.001), those during 6MWT had a statistically significant mean decrease of -0.171 (95%CI: -0.195, -0.148) (p-value &lt; 0.001), and those between the 2MMT and 6MWT had a statistically significant, mean difference of -0.226 (95%CI: -0.341, -0.111) (p-value &lt; 0.001). ### Conclusion We investigated the equivalence of the new cardiovascular endurance test using the 2MMT and 6MWT in healthy volunteers. These tests can provide valid estimates of VO2max in epidemiological studies. The 2MMT may be more relevant than expected. However, further studies are needed to determine the 2MMT’s sensitivity and MCID.