*3.3. Validity of the Reference Equation*

According to 20% of the sample (*n* = 31), no significant differences (*p* = 0.984) were observed between the actual values performed by participants (319 [141; 360] steps) and those obtained by the equation (290 [173; 339] steps).

#### **4. Discussion**

This study determined a reference equation for IST performance and showed that the variability of results was explained by sex and age. It is important to mention that these values were obtained from participants over a wide range of ages between 19 and 93 years. The inclusion of age as a determinant variable for the variability of results was expected because the aging process causes skeletal muscle contractile function loss [29] and lower oxygen consumption [30], leading to a worse exercise capacity. Another explanation for these results is the greater probability of the difficulty for older adults to negotiate stairs as a marker of functional decline, which can influence the performance in step mode testing. This difficulty is not only associated with reduced lower-limb strength, but also with reduced sensation and balance, and an increased fear of falling [31]. In fact, age has been the most indicated predictor of performance in exercise field tests, namely in other steps tests [32–34], walking tests [16,17,35], and upper-limb exercise tests [17].

We also expected that sex could influence the performance of the IST. Sex is considered a strong predictor of exercise capacity [36] that is consistently observed in reference equations for the prediction of exercise capacity in field tests, especially in walking tests [35,37,38] and step tests [32–34].

On the other hand, we expected that other independent variables could influence the performance of the IST, highlighting body composition. Body composition, through anthropometric measures, can be a determinant variable in the assessment of exercise capacity [39]; however, the variables used in our study (weight, height, and BMI) did not influence performance in the IST. We hypothesized that weight could be a predictor of a smaller number of steps performed. Overweight, associated with more fat accumulation, increases the workload on horizontal (walking) and vertical displacements, which normally occur during step mode testing [40–42]. Despite the controversy over whether BMI is the best measure of obesity, BMI ranges are still based on excess body fat [28]. As such, it was expected that this variable also could negatively influence performance in terms of the number of step tests; however, this was not observed in our reference equation. The lack of variability in BMI values in our sample was not a limitation for this observation because the participants included in the development of the reference equation (80% of participants) presented a wide variety of BMI values (minimum of 17 and maximum of 37 kg/m2). Although it could be argued that the inclusion of patients with a lower (≤18.5 kg/m2: underweight) and higher ( ≥ 30 kg/m2: obese) BMI introduced a bias because reference values should be derived from apparently healthy individuals, the main aim of our study was to achieve maximum representativeness from community-dwelling people. There is no consensus in the literature regarding the inclusion of participants with the lowest and highest BMI values for the determination of reference equations for field test where these participants had been excluded from some studies [32,35,38] but not from others [33,43].

The absence of differences between the number of steps achieved by participants and those predicted by the equation in 20% of participants of our sample is considered a strength in our study, suggesting that this equation is valid and can be applied in clinical practice. Another important strength was the accomplishment of the sample size calculated prior to the study, despite the restrictions due to the COVID-19 pandemic during data collection. Additionally, this equation was developed using only age and sex variables, facilitating its direct translation to clinical practice. However, the inclusion of other variables, such as peripheral muscle strength, to explore their influence in the performance of the IST is important in future studies. Additionally, in future studies, a larger sample size is important to determine normative values for the IST, contributing to greater information on its clinical interpretability.

This study has limitations that are important to mention. Firstly, the use of a convenience sample might have affected the results. More efforts are necessary to recruit participants from different settings and geographical locations to obtain a representative sample because our data collection was only performed in the north of Portugal. Despite the results from this study being obtained from participants over a wide range of between 19 and 93 years of age, it is important to mention that the number of participants in each age decade was not proportional, in which a lower number of participants were observed in the older decades. More participants in older decades are equally important, especially for the determination of normative values.

This is the first study to develop a reference equation for the IST in the Portuguese adult population and, to the best of our knowledge, one of the only studies to determine a national reference equation based on the performance of a step test (number steps). In fact, in the literature, most of the reference equations for step tests are developed with the intent to predict cardiorespiratory fitness, based on the estimation of maximum oxygen uptake [1,44–46]. Equations based on the performance of field tests provide advantages in clinical practice, yielding the utility of these tests as an outcome measure of exercise capacity. In addition, they provide an easy interpretation of patients' exercise capacity and prognosis in different conditions/diseases.

#### **5. Conclusions**

The established reference equation for the IST demonstrated that age and sex were the determinant variables for the variability of the results. This study also demonstrated that there were no differences between the actual values performed by participants and those obtained by the equation. These results will help to detect people with a lower exercise capacity, yielding the development of exercise programs and the assessment of their effectiveness.

**Author Contributions:** Conceptualization, R.V. and A.M.M.; methodology, R.V., C.C. and A.M.M.; software, R.V.; validation, C.C. and A.M.M.; formal analysis, R.V., C.C. and A.M.M.; investigation, R.V., A.T., C.S., F.M., L.T., L.M., T.G., C.M., C.C. and A.M.M.; resources, C.C.; data curation, R.V.; writing—original draft preparation, R.V., A.T., C.S., F.M., L.T. and L.M.; writing—review and editing, C.M., C.C. and A.M.M.; visualization, C.C. and A.M.M.; supervision, C.C. and A.M.M.; project administration, R.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the School of Health—Polytechnic Institute of Porto (protocol code E0134, date of approval: 13 April 2020). The study was registered at ClinicalTrials.gov (registry number NCT04801979).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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