Treatment simulation is usually performed with a conventional simulator using kV X-rays or with a computed tomography (CT) simulator before the treatment course begins. The purpose is to verify patient setup under the same conditions as for treatment planning. Systematic (preparation) setup errors can be introduced by this process. The purpose of this study is to characterize the setup errors using electronic portal image (EPI) analyses and to propose a method to reduce the systematic component by performing simulation and patient preparation on the treatment machine. In this study, the first four or five days EPIs were analyzed from a total of 533 prostate cancer patients who were simulated on conventional simulators. We characterized setup errors using four parameters: {M(microi), Sigma (microi), RMS(microi), sigma (sigmai)}, where microi and sigmai are individual patient mean and standard deviation, M, Sigma, and RMS are the mean, standard deviation, and root-mean-square of underlying variables (microi and sigmai). We have performed a simulation of removing systematic components by correcting the first day setup error. As a comparison, we also carried out a similar analyses for patients simulated on a CT simulator and patients treated on a linac with an on-board kV CT imaging system, although a limited number of patients were available in these two samples. We found that Sigma (/ui)=(2.6,3.4,2.4) mm, and RMS(sigmai)=(1.5,1.9,1.0) mm in lateral, anterior/posterior, and cranial/caudal directions, indicating that systematic errors are much larger than random errors. Strong correlations were found between measurement on the first day and microi, implying the first day's measurement is a good predictor for microi. The same parameters were also computed for days 2-4, with and without the first day correction. Without correction, M(microi)2-4=(0.7,1.6,-1.0) mm, and Sigma(microi)2-4=(2.6,3.5,2.4) mm. With correction, M(microi)2-4=(0.0,0.4,0.4) mm, much closer to zero, and Sigma(microi)2-4=(1.8,2.2, 1.2) mm, also much smaller. While the use of a CT simulator can reduce the systematic errors, the benefits of first day correction can still be observed, although at a smaller magnitude. Therefore, the systematic setup error can be significantly reduced if the patient is marked and fields are verified on the treatment machine on the first fraction, preferably with an on-board kV imaging system.