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Occupational stress has in a large number of studies been proven to increase and challenge employees’ sustained employability. By optimizing workers’ health surveillance programs (WHS), predictions of future health can be made and prevention of mental health illness can be facilitated. Current WHS include mainly subjective measures of mental health and stress but validity of these measures is frequently insufficient. The objective of this study was to explore the relation between heart rate variability (HRV) and the work ability index (WAI), and therefore contribute to more effective WHS, and the promotion of sustained employability. The study was designed as cross-sectional, and a linear regression analysis was applied with WAI as the dependent variable and the HRV measurements separately (SDNN, RMSSD, MHRR and normalized coherence) as independent variables. Work ability was measured with the index WAI, short version (WAI-Netzwerk, 2012). HRV was measured with the biofeedback pulse sensor tool emWave Pro (Institute of HeartMath), using the six-breath protocol. The result showed that one of the HRV measures mean heart rate range (the difference between the maximum and minimum heart rate during each breathing cycle) was negatively associated with WAI, when controlled for age B = -.26, 95% CI [-.48, -.05], β = -.38, t = -2.48, p = .016. The data gave unexpected results, and the conclusion cannot be made that HRV is an accurate and objective measure for work ability and sustained employability. Future studies should investigate other constructs associated with sustainable employability and continue to find evidence for HRV as an objective measure of mental health.