Subjects
We recruited subjects aged 60–85 to participate in this cross-sectional study and collected data over a 59-month period from December 2013 to October 2018. We obtained verbal informed consent from all subjects prior to the trial and the Ethics Committee of Wenzhou People’s Hospital approved the design of the study. Before recruiting the subjects, the researchers informed the subjects about the purpose of the study and asked them to fill out a questionnaire to ensure the health of the subjects. The researchers also verified the questionnaire before the subjects performed the 6MWT.
The criteria for inclusion were as follows: healthy subjects, subjects with 18 kg/m2≤ body mass index (BMI) <30 kg/m2 and subjects aged 60–85 years. The exclusion criteria included pre-existing limitations in exercise capacity (muscle, joint or nerve intervention), basic systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, basic heart rate <50 bpm or ≥100 bpm, any organic disease, abnormal lung function (FEV1< 80% predicted or FEV1/FVC < 70%), extreme weight (BMI <18 kg/m2 or BMI >30 kg/m2) and respiratory symptoms during the past 1 month.
Procedure
Subjects were asked to avoid alcohol, caffeine and meals for at least two hours prior to testing, as well as strenuous exercise within 24 hours prior to testing. The age, height, weight, BMI and blood pressure of the subjects were measured prior to testing. Age was verified by the subject’s identity card. An altimeter was used to measure the subject’s height without shoes when their back was straight. Body weight (kg) was measured using an electronic weight scale, and the BMI of the subjects was calculated as weight/height2 (expressed in kg/m2). Pulmonary function was measured using a standard portable pulmonary function meter (Vitalograph Alpha, Ireland) according to ATS recommendations. [15]
Questionnaire assessment
The health state of the participants was assessed by a standardized questionnaire that included: “Are you healthy?”, “Do you have high blood pressure, diabetes or other chronic disease?”, “Are you taking medication regularly?”, and “Do you have a problem with walking or do you need a walking aid when you walk?”. In addition, we also asked the subjects whether they participated in physical activities. The questions were as follows: “How many times do you participate in physical activity in a week? How long does it take?”. If the subject had performed lower extremity exercises for at least 20 minutes 3 times per week in the last 4 weeks, they were classified as “physically active subjects”. [16] In contrast, the subjects were classified as “sedentary subjects” if they did not meet the above criteria.
Six-minute walk test
The 6MWT was conducted in accordance with ATS standards [4]. The 6MWT was completed in a straight, long, flat, closed, 30-meter corridor that people rarely pass. The researchers made a mark on the corridor every 3 meters. An orange traffic cone was placed in the corner of the corridor as a sign. The starting line represented the start and end points of every 60 meters and was marked with colorful tape. To reduce the biological rhythm and temperature effects, the 6MWT was completed between 9:00 am and 16:00 pm, and the temperature range was between 20 °C and 25 °C. Subjects were asked to sit in a chair for at least 10 minutes before the 6MWT. The researchers measured and recorded the resting blood pressure, heart rate, and oxygen saturation. Subjects were informed that the purpose of this study was to measure the distance they walked quickly within the specified time. The subjects were then asked to walk in the corridor as quickly as possible within six minutes. If the subject had chest pain, leg cramps, dizziness, or difficulty breathing, they could stop and rest for a while, but we encouraged them to continue walking as soon as they recovered. The 6MWT was monitored by a specific investigator who used an electronic timer and a 30 meter long meter scale to record the time and distance. The subjects received a standardized encouragement every 30 seconds (i.e., “You’re doing well, stick to it” and “Do your best”). The distance covered over the six minutes was recorded as the 6MWD. We immediately measured the blood pressure, heart rate and oxygen saturation of the subjects at the end of the 6MWT. After the test was completed, the modified Berger’s Dyspnea Scale (MBD) was used to assess the subject’s degree of dyspnea. The MBD scale ranges from 0 (no dyspnea) to 10 points (maximum dyspnea). The subjects completed two 6MWTs monitored by the same operator. The second 6MWT was completed in 2 hours and the longer 6MWD was used for further analysis.
Data analysis
The Kolmogorov-Smirnov test was used to assess the normality of the data. Data are expressed as the mean ± standard deviation (SD), number and percentage, as appropriate. Descriptive analysis methods were used to analyze the characteristics of the subjects. The paired sample Student’s t-test was used to compare activity and gender among two groups, and the unpaired samples Student’s t-test was used to compare two 6MWDs. The paired t-test was used to compare the measured 6MWD in our subjects and the 6MWD based on previously published reference equations derived from foreign studies. [6, 11, 12, 17–19] The repeatability of the two 6MWTs was tested by the intraclass correlation coefficient (ICC) and Bland Altman analysis. [20] We first used univariate analysis with the Spearman’s correlation test to evaluate the correlation between the 6MWD and the variables, and then used a forward stepwise multiple regression analysis to establish the reference equations for the 6MWD. The most significant categorical variable was added to the model at each step, and the process continued until no other statistically significant variables could be added. A p-value >0.05 was used to determine whether to enter and reject variables. Data analysis was performed on Windows statistical software using SPSS (version 17.0; SPSS, Inc., Chicago, IL). In all analyses p values <0.05 were considered significant.