Placebo effects can be induced by learning and conditioning processes, which in turn are influenced and modulated by glucocorticoids. Accordingly, previous research has shown that intervention-related associative learning can be modulated through exogenous as well as endogenous glucocorticoids. Thus, the aim of this study was to elucidate whether placebo effects induced by conditioning is modulated by daily fluctuations of endogenous cortisol levels in healthy male and female subjects. Overall 77 participants underwent a two-phased placebo conditioning paradigm for pain analgesia. Subjects were randomized in two groups, which underwent placebo preconditioning either in the morning (08:00-10:00, i.e. with high endogenous cortisol levels) or in the afternoon (16:00-18:00, i.e. with low endogenous cortisol levels). Placebo effects were assessed two days later at noontime (12:00-13:00), with possible differences between groups as an indicator of glucocorticoid modulation on the placebo learning. Results indicated a significant conditioned placebo-induced analgesia, resulting in a placebo effect of small to medium size. Cortisol levels on conditioning day significantly differed between groups and cortisol levels were similar during assessment of placebo effects. Groups did not differ in their mean reduction in pain sensation, thus the placebo effect was not affected by differences in cortisol levels during the conditioning of placebo effects. The present study does not indicate a moderation of placebo conditioning by endogenous glucocorticoid levels.