Many studies on stress and pain rely, solely or mainly, on plasma cortisol assessment. Confounding factors, such as handling, may cause a release of cortisol making the interpretation of the results difficult. We looked at the influence of duration of restraint on the plasma cortisol levels of one-to-two month old calves. Forty-three calves were divided into four groups according to the interval between restraint and blood sampling: i) Group 0, immediate blood-sampling; ii) Group 0.5M, half a minute restraint; iii) Group 1M, one minute restraint and iv) Group 2M, two minutes restraint. The only increase in plasma cortisol, compared with all the other groups, was seen with blood sampling after two minutes of restraint. This study provides evidence to suggest that cortisol released as a result of handling stress is not evident if blood sampling is carried out within one minute of restraining calves.