Susceptibility to heat illness during physically demanding work in hot environments is greater on the second of two consecutive workdays. While it has been demonstrated that heat storage is exacerbated on the second compared to first workday in older workers (50-65?yr), the effects on heart rate variability (HRV), an established surrogate of cardiac autonomic modulation, remain unclear. This study evaluated HRV in older workers on the day following prolonged work in the heat. Electrocardiogram was recorded in nine older (53-64?yr) males at rest, during three 30-min bouts of semi-recumbent cycling at fixed rates of metabolic heat production (150, 200, 250?W/m(2)), each separated by 15-min recovery. Experiments were conducted in hot-dry conditions (40?°C, 20% relative humidity), immediately prior to (Day 1), and on the day following (Day 2), a prolonged work simulation (?7.5?hr) involving moderate intensity intermittent exercise in hot-dry conditions (38?°C, 34% relative humidity). Core temperature, as well as time, frequency, and nonlinear HRV indices were derived for analysis during rest, the final 5-min of exercise at the highest heat production and recovery. The change in core temperature at the end of work (mean?±SD) was significantly greater on Day 2 (1.0?°C?±0.3) relative to Day 1 (0.8?°C?±0.2; p??0.05). Prolonged work in the heat did not modulate next-day heart rhythms, as reflected by HRV, despite augmented core temperature. While HRV can reflect physiological aspects of cardiac autonomic stressors, these findings indicate it does not provide a means to identify exacerbated heat strain in older workers over consecutive work shifts in the heat.