The combination of analgesic drugs with different pharmacological properties may show better efficacy with less side effects. Aim of this study was to examine the analgesic and antihyperalgesic properties of the weak opioid tramadol and the non-opioid acetaminophen, alone as well as in combination, in an experimental pain model in humans. After approval of the local Ethics Committee, 17 healthy volunteers were enrolled in this double-blind and placebo-controlled study in a cross-over design. Transcutaneous electrical stimulation at high current densities (29.6 ± 16.2 mA) induced spontaneous acute pain (NRS = 6 of 10) and distinct areas of hyperalgesia for painful mechanical stimuli (pinprick-hyperalgesia). Pain intensities as well as the extent of the areas of hyperalgesia were assessed before, during and 150 min after a 15 min lasting intravenous infusion of acetaminophen (650 mg), tramadol (75 mg), a combination of both (325 mg acetaminophen and 37.5 mg tramadol), or saline 0.9%. Tramadol led to a maximum pain reduction of 11.7 ± 4.2% with negligible antihyperalgesic properties. In contrast, acetaminophen led to a similar pain reduction (9.8 ± 4.4%), but a sustained antihyperalgesic effect (34.5 ± 14.0% reduction of hyperalgesic area). The combination of both analgesics at half doses led to a supra-additive pain reduction of 15.2 ± 5.7% and an enhanced antihyperalgesic effect (41.1 ± 14.3% reduction of hyperalgesic areas) as compared to single administration of acetaminophen. Our study provides first results on interactions of tramadol and acetaminophen on experimental pain and hyperalgesia in humans. Pharmacodynamic modeling combined with the isobolographic technique showed supra-additive effects of the combination of acetaminophen and tramadol concerning both, analgesia and antihyperalgesia. The results might act as a rationale for combining both analgesics.