This experiment aimed to investigate the efficacy of twice-daily, nonconsecutive heat acclimation (TDHA) in comparison to once-daily heat acclimation (ODHA) and work matched once- or twice-daily temperate exercise (ODTEMP, TDTEMP) for inducing heat adaptations, improved exercise tolerance, and cytokine (immune) responses. Forty males, matched biophysically and for aerobic capacity, were assigned to ODHA, TDHA, ODTEMP, or TDTEMP. Participants completed a cycling-graded exercise test, heat acclimation state test, and a time to task failure (TTTF) at 80% peak power output in temperate (TTTFTEMP : 22°C/40% RH) and hot conditions (TTTFHOT : 38°C/20% RH), before and after 10-sessions (60 min of cycling at ~2 W·kg-1 ) in 45°C/20% RH (ODHA and TDHA) or 22°C/40% RH (ODTEMP or TDTEMP). Plasma IL-6, TNF-α, and cortisol were measured pre- and postsessions 1, 5, and 10. ODHA and TDHA induced equivalent heat adaptations (P 0.05) following ODHA (+14 ± 4%), TDHA (14 ± 8%), ODTEMP (9 ± 10%) or TDTEMP (8 ± 13%). Acute (P 0.05) increases were observed in IL-6, TNF-α, or cortisol during ODHA and TDHA, or ODTEMP and TDTEMP. Once- and twice-daily heat acclimation conferred similar magnitudes of heat adaptation and exercise tolerance improvements, without differentially altering immune function, thus nonconsecutive TDHA provides an effective, logistically flexible method of HA, benefitting individuals preparing for exercise-heat stress.