The submarine working and living environment is an isolated, confined, and extreme (ICE) environment where a continuous on-watch is required to fulfill the tactical objectives. The current study examined whether a physiological and behavioral adjustment to an operational watch standing scheme occurred in terms of hormonal secretion (i.e., melatonin and cortisol) and sleep during a 67-days undersea mission. The crew followed a strict scheme of watch-on blocks at 0:00-06:00 h and at 12:00-18:00 h (group 1, diurnal sleep group) or watch-on blocks at 06:00-12:00 h and 18:00-24:00 h (group 2, nocturnal sleep group). We sampled saliva during the operational blocks over a 24h period at day 55 of the mission and collected sleep actigraphy data during the entire mission in 10 participants. Sleep showed a biphasic split pattern with significantly unequal distributions of total sleep time (TST) and sleep efficiency (SE) between the two sleeping blocks, i.e., one long and one short sleep bout. Melatonin secretion showed no adjustment at the end of the mission to the watch standing blocks, following an endogenous circadian rhythm independent from the social zeitgebers with indications of a phase shift. Cortisol secretion however matched the biphasic work-sleep shift routine. Human physiology does not fully obey operational needs and there are differences in adjustment between melatonin and cortisol. A watch standing schedule that provides a balance between physiology and operationality still needs to be established. The potential adaptation effects of bright light therapy and melatonin supplementation should be investigated in future research.