Introduction Although Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection worldwide, little is known about the natural course of the bacterial load during infection. We investigated the natural course of the bacterial load in the interval between screening and returning for treatment in genital and anorectal CT-infections. Materials & Methods CT-positive patients, visiting our STI-clinic in the Netherlands from June 2011-January 2014, provided a second urogenital and/or anorectal sample when returning for treatment (diagnostic sample = T1; treatment sample = T2). Patient-record provided data about the days between samples and the date of last unsafe sex. Included patients were >= 18 years old, HIV-negative and did not report antibiotic use in the study-interval. CT load was quantified using qPCR. CT load was log-transformed, and a CT load difference (Delta-CT load) of > 1 log was deemed clinically relevant. Chi-square test compared load category distributions over time (decrease/equal/increase), between sample types. Results 274 patients provided 296 paired samples. Majority of samples had a stable CT load in the interval T1-T2 (66.3%, 73.1% and 48.6% for vaginal swabs, urine and anorectal swabs resp. p = 0.07). Load decreased in 17-41% of patients, while +/- 10% of patients showed an increase in CT load. No association between Delta-CT load and the interval T1-T2 was observed. Large variations can be seen in CT load at T1 and over time. Discussion The majority (+/- 90%) of patients have a stable or decreasing CT load in the time interval between screening and returning for treatment. The number of days between sampling was not associated with change in CT load. In the first month after the last unsafe sex, only stable CT loads were seen. Our data seems to indicate that when most patients visit an STI-clinic, recommended 2 weeks after infection, the infection has already been established or is in its downward phase.