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In Europe, <i>Ixodes ricinus</i> plays a major role as a vector of <i>Borrelia burgdorferi</i> sensu lato (s.l.) spirochaetes, the causative agents of Lyme borreliosis, among other pathogens. In unfed ticks, <i>Borrelia</i> spirochaetes experience prolonged nutrient restriction. However, only few studies exist with regard to <i>Borrelia</i> infections in unfed ticks of different physiological ages. Changing body dimensions of unfed ticks, due to the consumption of energy reserves, allow physiological age estimation. The present study investigated the relationship of morphometric age with <i>Borrelia</i> prevalence and spirochaete load in 1882 questing <i>I. ricinus</i> nymphs, collected at two different locations in northern Germany in 2020. In addition, <i>Borrelia</i> species composition was investigated by employing a reverse line blot (RLB) probe panel suitable for the detection of ten different <i>B. burgdorferi</i> s.l. species, as well as the relapsing-fever spirochaete <i>B. miyamotoi</i>. Overall, <i>Borrelia</i> prevalence was 25.8% (485/1882). Whilst there was no statistically significant difference in <i>Borrelia</i> prevalence between the different morphometric age groups, <i>Borrelia</i> infection intensity as determined by probe-based quantitative real-time PCR significantly declined with increasing morphometric age. <i>Borrelia</i> species differentiation by RLB was successful in 29.5% of positive ticks, and revealed <i>B. afzelii</i> as the dominating species (65.0% of the differentiated infections). Additionally, <i>B. garinii</i>, <i>B. valaisiana</i>, <i>B. burgdorferi</i> sensu stricto, <i>B. spielmanii</i>, and <i>B. miyamotoi</i> were detected.