Laboratory animals are frequently subjected to routine procedures, such as injections or the withdrawal of blood samples. Acute stress caused by such procedures is associated with physiological changes that can have a strong impact on experimental results. This study investigated the integrated effects of cage enrichment, social housing and handling on the acute stress response of animals subjected to routine experimental procedures. Female mice of two inbred strains (BALB/c and C57BL/6) were housed under either minimal husbandry conditions (MH: no cage enrichment, infrequent handling and a period of individual housing) or enriched husbandry conditions (EH: with cage enrichment, frequent handling and social housing at all times). One mouse in each cage was implanted with a radio-telemetry transmitter for measuring heart rate (HR) and body temperature (BT). The animals were subjected to intraperitoneal injections or short periods of restraint. In addition to telemetry measurements, thymus weight and tyrosine hydroxylase (TH) activity were assessed. It was found that individual housing under MH conditions, as compared with social housing under EH conditions, elevated both basal HR and BT, and significantly elevated the relative recovery time following routine experimental procedures. Thymus weight and TH activity suggested a long-term stress response under MH conditions following individual housing, although the influence of transmitter implantation and (repeated) acute stress remains to be investigated. The results emphasise that husbandry conditions should be taken into account when evaluating physiological measures after routine procedures.