Stressful manipulation by humans has been previously shown to result in opening the pre-orbital gland of the newborn red deer. We hypothesized that exposure of red deer to stressful handling would result in opening the pre-orbital gland. Further, we tested the potential factors associated with pre-orbital opening, including season, sex, age and struggling behavior. Pre-orbital gland status was observed in 76 red deer (48 males, 28 females) during 281 handling events with the animal isolated and fixed in a mechanical restraint cradle (crush) within 3 consecutive years. The deer age ranged from 1 to 5 yr in males, and from 1 to 11 yr in females at the beginning of observations. The proportion of handling events with an open gland was considerably greater during than before handling (27.76 vs. 0%, respectively, P < 0.001). The deer that struggled or stiffened revealed 2-fold greater incidence of pre-orbital opening (P < 0.01), regardless of sex. Thus, pre-orbital opening seemed to accompany the behaviors that have been found to be related to acute stress. The probability that the animal opens its pre-orbitals during handling sharply increased at the beginning of the rut (mating season, P < 0.01). Deer that threatened the handler (10% out of 215 observations) opened their pre-orbitals 30 times more often than nonthreatening ones. These effects indicate that other motivations are likely involved in the opening of pre-orbitals, in addition to the perception of a stressful event. Incidence of pre-orbital opening decreased with age in both sexes (P < 0.05) across 3 consecutive yr of observations, which suggests habituation to repeated handling. Sex of the animal did not influence the probability of pre-orbital opening. We found significant variability in pre-orbital opening across the individuals (P < 0.001). In conclusion, we confirmed an association between opening of the pre-orbital gland and stressful handling in sub-adult and adult red deer, although repeated human handling did not elicit such incidence of pre-orbital opening as found in newborn red deer calves. Our results support multifactorial origins of pre-orbital opening and prompt the necessity of further research to distinguish between different motivations that might have been involved in opening of pre-orbitals.