The 2-minute step test (2MST) requires minimal resources and is valid in adults older than 60 years. Here we investigate if the 2MST can be used to estimate peak oxygen consumption (VO[subscript 2]peak) in healthy middle-aged adults (40-59 years old). Thirty-six participants completed the 2MST and a maximal graded treadmill test with breath-by-breath gas analysis. Outcome measures included the number of steps completed in the 2MST, VO[subscript 2]peak (ml/kg/min), waist circumference (cm), resting blood pressure (mmHg) and body mass index (kg/m[superscript 2]). Data were analyzed using Pearson correlation coefficients and multivariate linear regression analyses. There was a moderate relationship between 2MST peak steps completed and VO[subscript 2]peak (r = 0.54, p = 0.001). The 2MST model, including 2MST peak steps, height and waist circumference, predicted VO[subscript 2]peak with r = 0.80 (p < 0.001). The 2MST may be a good predictor of VO[subscript 2]peak in healthy middle-aged adults, and may be suitable for primary health-care settings and large-scale epidemiological studies. Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12617000091303, http://www.ANZCTR.org.au/ACTRN12617000091303.aspx, date registered: 17 January 2017.